Ancient and Recent Hybridization in the Oreochromis Cichlid Fishes.

Cichlid fishes of the genus Oreochromis (tilapia) are among the most important fish for inland capture fisheries and global aquaculture. Deliberate introductions of non-native species for fisheries improvement and accidental escapees from farms have resulted in admixture with indigenous species. Such hybridization may be detrimental to native biodiversity, potentially leading to genomic homogenization of populations and the loss of important genetic material associated with local adaptation. By contrast, introgression may fuel diversification when combined with ecological opportunity, by supplying novel genetic combinations. To date, the role of introgression in the evolutionary history of tilapia has not been explored. Here we studied both ancient and recent hybridization in tilapia, using whole genome resequencing of 575 individuals from 23 species. We focused on Tanzania, a natural hotspot of tilapia diversity, and a country where hybridization between exotic and native species in the natural environment has been previously reported. We reconstruct the first genome-scale phylogeny of the genus and reveal prevalent ancient gene flow across the Oreochromis phylogeny. This has likely resulted in the hybrid speciation of one species, O. chungruruensis. We identify multiple cases of recent hybridization between native and introduced species in the wild, linked to the use of non-native species in both capture fisheries improvement and aquaculture. This has potential implications for both conservation of wild populations and the development of the global tilapia aquaculture industry.

Salinity-responsive histone PTMs identified in the gills and gonads of Mozambique tilapia (Oreochromis mossambicus).

BACKGROUND: Histone post-translational modifications (PTMs) are epigenetic marks that can be induced by environmental stress and elicit heritable patterns of gene expression. To investigate this process in an ecological context, we characterized the influence of salinity stress on histone PTMs within the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus). A total of 221 histone PTMs were quantified in each tissue sample and compared between freshwater-adapted fish exposed to salinity treatments that varied in intensity and duration. RESULTS: Four salinity-responsive histone PTMs were identified in this study. When freshwater-adapted fish were exposed to seawater for two hours, the relative abundance of H1K16ub significantly increased in the gills. Long-term salinity stress elicited changes in both the gills and testes. When freshwater-adapted fish were exposed to a pulse of severe salinity stress, where salinity gradually increased from freshwater to a maximum of 82.5 g/kg, the relative abundance of H1S1ac significantly decreased in the gills. Under the same conditions, the relative abundance of both H3K14ac and H3K18ub decreased significantly in the testes of Mozambique tilapia. CONCLUSIONS: This study demonstrates that salinity stress can alter histone PTMs in the gills and gonads of Mozambique tilapia, which, respectively, signify a potential for histone PTMs to be involved in salinity acclimation and adaptation in euryhaline fishes. These results thereby add to a growing body of evidence that epigenetic mechanisms may be involved in such processes.

Hypogonadotropic hypogonadism in male tilapia lacking a functional rln3b gene.

Relaxin 3 is a neuropeptide that plays a crucial role in reproductive functions of mammals. Previous studies have confirmed that rln3a plays an important role in the male reproduction of tilapia. To further understand the significance of its paralogous gene rln3b in male fertility, we generated a homozygous mutant line of rln3b in Nile tilapia. Our findings indicated that rln3b mutation delayed spermatogenesis and led to abnormal testes structure. Knocking out rln3b gene resulted in a decrease in sperm count, sperm motility and male fish fertility. TUNEL detection revealed a small amount of apoptosis in the testes of rln3b-/- male fish at 390 days after hatching (dah). RT-qPCR analysis demonstrated that mutation of rln3b gene caused a significant downregulation of steroid synthesis-related genes such as cyp17a1, cyp11b2, germ cell marker gene, Vasa, and gonadal somatic cell marker genes of amh and amhr2. Furthermore, we found a significant down-regulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes, while a significantly up-regulation of the dopamine synthetase gene in the rln3b-/- male fish. Taken together, our data strongly suggested that Rln3b played a crucial role in the fertility of XY tilapia by regulating HPG axis genes.

Coping with salinity extremes: Gill transcriptome profiling in the black-chinned tilapia (Sarotherodon melanotheron).

Steeper and sometimes extreme salinity gradients increasingly affect aquatic organisms because of climate change. Hypersalinity habitats demand powerful physiological adaptive strategies. Few teleost species have the capacity to spend their whole life cycle in salinities way over seawater levels. Focusing on the multifunctional gill, we unraveled the tilapia S. melanotheron key strategies to cope with different environmental conditions, ranging from freshwater up to hypersaline habitats. De novo transcriptome assembly based on RNAseq allowed for the analysis of 40,967 annotated transcripts among samples collected in three wild populations at 0, 40 and 80 ‰. A trend analysis of the expression patterns revealed responses across the salinity gradient with different gene pathways involved. Genes linked to ion transport, pH regulation and cell surface receptor signaling were mainly upregulated in the high salinity habitat. We identified tight junction proteins that were critical in high salinity habitats and that were different from the well-known tightening junctional proteins identified and expressed in fresh water. Expression profiles also suggest a change in the vascular tone that could be linked to an osmorespiratory compromise not only in fresh water, but also in high salinity environments. A striking downregulation of genes linked to the immune system and to the heat shock response was observed suggesting an energetic trade-off between immunity and acclimation/adaptation in the hypersaline habitat. The high expression of transcripts coding for immune and heat shock response in the freshwater habitat suggests the establishment of powerful mechanisms to protect gills from environmental threats and to maintain protein integrity. Non-directional expression trends were also detected with an upregulation of genes only in the hypersaline habitat (80 ‰) or only in the marine habitat (40 ‰). Unravel physiological strategies in S. melanotheron populations will help to better understand the molecular basis of fish euryhalinity in salinity-contrasted environments.

Multiple trait comparison and global intestine transcriptional provide new insights into bases of heterosis in hybrid tilapia (Oreochromis niloticus × Oreochromis aureus).

Heterosis has been utilized in aquaculture for many years, yet its molecular basis remains elusive. Therefore, a comprehensive analysis of heterosis was conducted by comparing growth, digestion and biochemistry indices, as well as the intestinal gene expression profiles of Nile tilapia, blue tilapia and their hybrids. The results revealed that hybrid tilapia demonstrated an enhanced growth traits and elevated digestive enzyme activity compared to Nile and blue tilapia. Additionally, the hybrid tilapia displayed superior antioxidants and non-specific immune levels, with increased levels of catalase (CAT), alkaline phosphatase (AKP), acid phosphatase (ACP), glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (TAOC), lysozyme, and immunoglobulin M (IgM) relative to Nile and blue tilapia. Moreover, 3392, 2470 and 1261 differentially expressed genes (DEGs) were identified in the intestinal tissues when comparing Nile tilapia to blue tilapia, hybrid tilapia to blue tilapia, and hybrid tilapia to Nile tilapia. Upon classifying the differentially expressed genes (DEGs), non-additively expressed DEGs accounted for 68.1 % of the total DEGs, with dominant and over-dominant expressed DEGs comprising 63.7 % and 4.4 % in the intestines, respectively. These non-additively expressed DEGs were primarily associated with metabolic, digestive, growth, and developmental pathways. This enrichment enhances our comprehension of the molecular underpinnings of growth heterosis in aquatic species.

Effects of CRISPR/Cas9 targeting of the myo-inositol biosynthesis pathway on hyper-osmotic tolerance of tilapia cells.

Myo-inositol is an important compatible osmolyte in vertebrates. This osmolyte is produced by the myo-inositol biosynthesis (MIB) pathway composed of myo-inositol phosphate synthase and inositol monophosphatase. These enzymes are among the highest upregulated proteins in tissues and cell cultures from teleost fish exposed to hyperosmotic conditions indicating high importance of this pathway for tolerating this type of stress. CRISPR/Cas9 gene editing of tilapia cells produced knockout lines of MIB enzymes and control genes. Metabolic activity decreased significantly for MIB KO lines in hyperosmotic media. Trends of faster growth of the MIB knockout lines in isosmotic media and faster decline of MIB knockout lines in hyperosmotic media were also observed. These results indicate a decline in metabolic fitness but only moderate effects on cell survival when tilapia cells with disrupted MIB genes are exposed to hyperosmolality. Therefore MIB genes are required for full osmotolerance of tilapia cells.

Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Coxsackievirus and adenovirus receptor inhibits tilapia lake virus infection via binding to viral segment 8 and 10 encoded protein.

The global aquaculture industry of tilapia (Oreochromis niloticus) has been significantly impacted by the emergence of tilapia lake virus (TiLV). However, effective prevention and control measures are still not available due to a lack of unclear pathogenesis of TiLV. Our previous transcriptome found that coxsackievirus and adenovirus receptor (CAR) was in response to TiLV infection in tilapia. To explore the potential function of OnCAR, the effect of OnCAR on TiLV proliferation was analyzed in this study. The OnCAR open reading frame (ORF) sequence of tilapia was 516 bp in length that encoded 171 amino acids with an Ig-like domain and transmembrane region. The OnCAR gene showed widespread expression in all investigated tissues, with the highest levels in the heart. Moreover, the OnCAR gene in the liver and muscle of tilapia exhibited dynamic expression levels upon TiLV challenge. Subcellular localization analysis indicated that OnCAR protein was mainly localized on the membrane of tilapia brain (TiB) cells. Importantly, the gene transcripts, genome copy number, S8-encoded protein, cytopathic effect, and internalization of TiLV were obviously decreased in the TiB cells overexpressed with OnCAR, indicating that OnCAR could inhibit TiLV replication. Mechanically, OnCAR could interact with viral S8 and S10-encoded protein. To the best of our knowledge, OnCAR is the first potential anti-TiLV cellular surface molecular receptor discovered for inhibiting TiLV infection. This finding is beneficial for better understanding the antiviral mechanism of tilapia and lays a foundation for establishing effective prevention and control strategies against tilapia lake virus disease (TiLVD).

Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

Ultra-structure, genetic characterization and Immunological approach of fish borne zoonotic trematodes (Family: Heterophyidae) of a redbelly tilapia.

Fish-borne zoonotic trematodes (FBZT) are highly significant zoonotic trematodes that can infect humans by eating raw or undercooked fish harboring active metacercaria. In this investigation, FBZT was found in samples of widely cultivated redbelly tilapia (Tilapia zillii) obtained from the Fayum governorate. Encysted metacercaria (EMC) infection was identified in fish belonging to the heterophyid family morphologically. The prevalence of heterophyid EMC was 30.5%. EMC was identified and implemented in a subsequent study on domestic pigeons (Columba livia domestica) carried out to allow adult flukes of Pygidiopsis (P.) genata; P. summa; and Ascocotyle (A.) pindoramensis species in their small intestine. This study presents the first report that combines ultra-structure, molecular approach of three species of heterophyid flukes, ultra-structure using transmission electron microscope in P. genata, and the study of host immunological responses and associated cytokines during Pygidiopsis species infection of pigeons in Egypt. Using Quantitative Real-time PCR (qRT- PCR), the gene expression levels of six cytokines (IL-1, IL-2, IL-6, IL-10, IFN-γ and TGF-ß3) were assessed. The molecular confirmation of P. genata, P. summa, and A. pindoramensis have a registration in the GenBank under accession number MT672308.1, OR083433.1, and OR083431.1, respectively. Throughout the infection, the gut produced cytokines in considerably variable amounts. As a result of the Pygidiopsis species infection in pigeons, our data showed distinctive cytokine alterations, which could aid in figuring out the immunological pathogenesis and host defense mechanism against this infection. This study focused on different types of fish-borne trematodes, particularly the zoonotically important ones.

Tilapia, a good model for studying reproductive endocrinology.

The Nile tilapia (Oreochromis niloticus), with a system of XX/XY sex determination, is a worldwide farmed fish with a shorter sexual maturation time than that of most cultured fish. Tilapia show a spawning cycle of approximately 14 days and can be artificially propagated in the laboratory all year round to obtain genetically all female (XX) and all male (XY) fry. Its genome sequence has been opened, and a perfect gene editing platform has been established. With a moderate body size, it is convenient for taking enough blood to measure hormone level. In recent years, using tilapia as animal model, we have confirmed that estrogen is crucial for female development because 1) mutation of star2, cyp17a1 or cyp19a1a (encoding aromatase, the key enzyme for estrogen synthesis) results in sex reversal (SR) due to estrogen deficiency in XX tilapia, while mutation of star1, cyp11a1, cyp17a2, cyp19a1b or cyp11c1 affects fertility due to abnormal androgen, cortisol and DHP levels in XY tilapia; 2) when the estrogen receptors (esr2a/esr2b) are mutated, the sex is reversed from female to male, while when the androgen receptors are mutated, the sex cannot be reversed; 3) the differentiated ovary can be transdifferentiated into functional testis by inhibition of estrogen synthesis, and the differentiated testis can be transdifferentiated into ovary by simultaneous addition of exogenous estrogen and androgen synthase inhibitor; 4) loss of male pathway genes amhy, dmrt1, gsdf causes SR with upregulation of cyp19a1a in XY tilapia. Disruption of estrogen synthesis rescues the male to female SR of amhy and gsdf but not dmrt1 mutants; 5) mutation of female pathway genes foxl2 and sf-1 causes SR with downregulation of cyp19a1a in XX tilapia; 6) the germ cell SR of foxl3 mutants fails to be rescued by estrogen treatment, indicating that estrogen determines female germ cell fate through foxl3. This review also summarized the effects of deficiency of other steroid hormones, such as androgen, DHP and cortisol, on fish reproduction. Overall, these studies demonstrate that tilapia is an excellent animal model for studying reproductive endocrinology of fish.

Concentrations, distribution, and key influencing factors of antibiotic resistance genes and bacterial community in water and reared fish tissues in a typical tilapia farm in South China.

Although previous studies have investigated the occurrence of antibiotic resistance genes (ARGs) in aquaculture, few have monitored the concentrations and propagation of ARGs in biological tissues or investigated the key factors influencing their spread in aquaculture. This study investigated the concentration, propagation, and distribution of ARGs and bacterial communities in water sources, pond water, and tilapia tissues, and their key influencing factors, in a typical tilapia farm. ErmF, sul1, and sul2 were the dominant ARGs with high concentrations. The total concentrations of ARGs (TCAs) in tilapia tissues decreased in the following order: stomach > scales > intestine > gills (P < 0.05). Redundancy analysis and multiple linear regression revealed that suspended solids (SS) and chemical oxygen demand (COD) were positively correlated with the dominant ARGs ermF sul2, and the TCAs (P < 0.05); additionally, Chloroflexi and Bacteroidetes in tilapia aquaculture water were positively correlated with the dominant ARGs ermF and sul2, as well as the TCAs (P < 0.05). This study suggests that SS and COD were the key factors driving the distribution and spread of ARGs in tilapia aquaculture water. Additionally, Chloroflexi and Bacteroidetes were the key bacterial flora affecting the propagation of ARGs in tilapia aquaculture systems.

Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family.

Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ends and encode proteins with no known homologues, apart from the segment 1, which encodes an orthomyxo-like RNA-dependent-RNA polymerase core subunit. Here we show that segments 1-3 encode respectively the PB1, PB2 and PA-like subunits of an active heterotrimeric polymerase that maintains all domains found in the distantly related influenza polymerase, despite an unprecedented overall size reduction of 40%. Multiple high-resolution cryo-EM structures of TiLV polymerase in pre-initiation, initiation and active elongation states, show how it binds the vRNA and cRNA promoters and performs RNA synthesis, with both transcriptase and replicase configurations being characterised. However, the highly truncated endonuclease-like domain appears inactive and the putative cap-binding domain is autoinhibited, emphasising that many functional aspects of TiLV polymerase remain to be elucidated.

Nonadditive and allele-specific expression of ghrelin in hybrid tilapia.

Background: Interspecies hybridization is an important breeding method to generate fishes with heterosis in aquaculture. Using this method, hybrid Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (Oreochromis aureus, ♂) has been produced and widely farmed due to its growth and appetite superiorities. However, the genetic mechanism of these advanced traits is still not well understood. Ghrelin is a crucial gene that regulates growth and appetite in fishes. In the present study, we focused on the expression characteristics and its regulation of ghrelin in the hybrid. Results: The tissue distribution analysis showed that ghrelin was predominantly expressed in the stomach in the hybrid. Ghrelin was more highly expressed in the stomach in the hybrid and Nile tilapia, compared to blue tilapia, showing a nonadditive pattern. Two single-nucleotide polymorphism (SNP) sites were identified including T/C and C/G from the second exon in the ghrelin gene from Nile tilapia and blue tilapia. By pyrosequencing based on the SNP sites, the allele-specific expression (ASE) of ghrelin in the hybrid was assayed. The result indicated that ghrelin in the hybrid showed higher maternal allelic transcript ratios. Fasting significantly increased ghrelin overall expression at 4, 8, 12, 24, and 48 h. In addition, higher maternal allelic transcript ratios were not changed in the fasting hybrids at 48 h. The cis and trans effects were determined by evaluating the overall expression and ASE values in the hybrid. The expression of ghrelin was mediated by compensating cis and trans effects in hybrid. Conclusion: In summary, the present lines of evidence showed the nonadditive expression of ghrelin in the hybrid tilapia and its regulation by subgenomes, offering new insight into gene expression characteristics in hybrids.

A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV).

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.

Genome editing in East African cichlids and tilapias: state-of-the-art and future directions.

African cichlid fishes of the Cichlidae family are a group of teleosts important for aquaculture and research. A thriving research community is particularly interested in the cichlid radiations of the East African Great Lakes. One key goal is to pinpoint genetic variation underlying phenotypic diversification, but the lack of genetic tools has precluded thorough dissection of the genetic basis of relevant traits in cichlids. Genome editing technologies are well established in teleost models like zebrafish and medaka. However, this is not the case for emerging model organisms, such as East African cichlids, where these technologies remain inaccessible to most laboratories, due in part to limited exchange of knowledge and expertise. The Cichlid Science 2022 meeting (Cambridge, UK) hosted for the first time a Genome Editing Workshop, where the community discussed recent advances in genome editing, with an emphasis on CRISPR/Cas9 technologies. Based on the workshop findings and discussions, in this review we define the state-of-the-art of cichlid genome editing, share resources and protocols, and propose new possible avenues to further expand the cichlid genome editing toolkit.

Comparative transcriptomes reveal different tolerance mechanisms to Streptococcus agalactiae in hybrid tilapia, nile tilapia, and blue tilapia.

Tilapia is one of the most economically important freshwater fish farmed in China. Streptococcosis outbreaks have been extensively documented in farmed tilapia species. Hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂) exhibit greater disease resistance than Nile tilapia (O. niloticus) and blue tilapia (O. aureus). However, the molecular mechanism underlying the enhanced tolerance of hybrid tilapia is still poorly understood. In this study, comparative transcriptome analysis was performed to reveal the different tolerance mechanisms to Streptococcus agalactiae in the three tilapia lines. In total, 1982, 2355, and 2076 differentially expressed genes were identified at 48 h post-infection in hybrid tilapia, Nile tilapia, and blue tilapia, respectively. Functional enrichment analysis indicated that numerous metabolic and immune-related pathways were activated in all three tilapia lines. The differential expression of specific genes associated with phagosome, focal adhesion, cytokine-cytokine receptor interaction, and toll-like receptor signaling pathways contributed to the resistance of hybrid tilapia. Notably, immune response genes in hybrid tilapia, such as P38, TLR5, CXCR3, CXCL12, PSTPIP1, and TFR, were generally suppressed under normal conditions but selectively induced following pathogen challenge. These results expand our knowledge of the molecular mechanisms underlying S. agalactiae tolerance in hybrid tilapia and provide valuable insights for tilapia breeding programs.

Sodium chloride alleviates oxidative stress and physiological responses induced by extreme winter cold in genetically improved farmed tilapia (GIFT; Oreochromis niloticus).

A 6-week trial was designed to investigate the effects of dietary sodium chloride supplementation on physiological, metabolic, and molecular stress response parameters. The findings showed that (1) there were no significant differences between sodium chloride supplementation groups (0.05S, 0.1S, and 0.15S) and the control group (P > 0.05), except for the 0.2S diet, which showed better final body weight, weight gain rate, specific growth rate, and feed conversion ratio than the control group (P < 0.05). (2) The hypothermic stress experiment results showed that the survival rates in the 0.1S and 0.15S diets were significantly higher than the control group (P < 0.05). (3) Transcription results showed that these enriched pathways in the gill were mainly energy metabolism and apoptosis pathways, while the major enrichment pathways in the liver were mainly amino acid metabolism and carbohydrate metabolism. (4) The plasma parameter results showed, compared to the control group, the 0.15S diet significantly increased the plasma GLU, TG contents, and Na+ and K+ concentrations and decreased the plasma ALT activity (P < 0.05). In addition, the 0.1S diet increased the plasma ALB content and Cl- concentration (P < 0.05). The gill Na+/K+-ATPase activity decreased markedly when the fish were fed the 0.1S and 0.15S diets (P < 0.05). The antioxidant enzyme activity results showed that the 0.1S and 0.15S diets significantly increased the T-SOD activities (P < 0.05). Gene expression results showed that compared to the control group, the 0.1S and 0.15S diets up-regulated the expression of gys, hsp70, mlcp, mlc, myosin, tnt mRNA, and down-regulated the akt, gk, and erk mRNA expression. Based on the regression analysis, the optimum dietary sodium chloride levels range from 0.10 % to 0.13 % of the diet, which could facilitate energy regulation, improve the immune response, and ultimately strengthen the cold resistance of GIFT.

The role of antifreeze genes in the tolerance of cold stress in the Nile tilapia (Oreochromis niloticus).

BACKGROUND: Tilapia is one of the most essential farmed fishes in the world. It is a tropical and subtropical freshwater fish well adapted to warm water but sensitive to cold weather. Extreme cold weather could cause severe stress and mass mortalities in tilapia. The present study was carried out to investigate the effects of cold stress on the up-regulation of antifreeze protein (AFP) genes in Nile tilapia (Oreochromis niloticus). Two treatment groups of fish were investigated (5 replicates of 15 fish for each group in fibreglass tanks/70 L each): 1) a control group; the fish were acclimated to lab conditions for two weeks and the water temperature was maintained at 25 °C during the whole experimental period with feeding on a commercial diet (30% crude protein). 2) Cold stress group; the same conditions as the control group except for the temperature. Initially, the temperature was decreased by one degree every 12 h. The fish started showing death symptoms when the water temperature reached 6-8 °C. In this stage the tissue (muscle) samples were taken from both groups. The immune response of fish exposed to cold stress was detected and characterized using Differential Display-PCR (DD-PCR). RESULTS: The results indicated that nine different up-regulation genes were detected in the cold-stressed fish compared to the control group. These genes are Integrin-alpha-2 (ITGA-2), Gap junction gamma-1 protein-like (GJC1), WD repeat-containing protein 59 isoform X2 (WDRP59), NUAK family SNF1-like kinase, G-protein coupled receptor-176 (GPR-176), Actin cytoskeleton-regulatory complex protein pan1-like (PAN-1), Whirlin protein (WHRN), Suppressor of tumorigenicity 7 protein isoform X2 (ST7P) and ATP-binding cassette sub-family A member 1-like isoform X2 (ABCA1). The antifreeze gene type-II amplification using a specific PCR product of 600 bp, followed by cloning and sequencing analysis revealed that the identified gene is antifreeze type-II, with similarity ranging from 70 to 95%. The in-vitro transcribed gene induced an antifreeze protein with a molecular size of 22 kDa. The antifreeze gene, ITGA-2 and the WD repeat protein belong to the lectin family (sugar-protein). CONCLUSIONS: In conclusion, under cold stress, Nile tilapia express many defence genes, an antifreeze gene consisting of one open reading frame of approximately 0.6 kbp.

Body yield and quality of fresh and post-freezing filet of Nile tilapia (Oreochromis niloticus) genetic groups.

The aim of this study was to evaluate the body yield and quality of fresh and post-freezing filet of male and female fish of inbred and non-inbred AquaAmérica genetic group and the hybrid between the AquaAmérica and Tilamax varieties. Forty fish (20 males and 20 females) of each genetic group were housed in four 48-m3 hapa net cages, getting 120 fish per cage. The fish were housed at 51 days of age and farmed for 269 days. Pre-slaughter weight was higher (P<0.05) in the AquaAmérica × Tilamax males (0.805±0.204 kg) than in the inbred AquaAmérica male (0.643±0.115 kg). Filet yield percentage was higher (P<0.05) in the AquaAmérica × Tilamax males (32.14±4.72%) than in the inbred AquaAmérica (28.15±2.67%) and non-inbred AquaAmérica (29.06±2.80%) males. Head and viscera yield percentages, pH, color values (L*, a* and b*), shear force, drip loss and cooking loss did not differ significantly between the genetic groups and sexes. Alterations in meat quality were observed after freezing. In conclusion, inbreeding in the AquaAmérica variety resulted in reduced slaughter weight for males; AquaAmérica × Tilamax males have a higher filet yield; and filet quality is not influenced by crossing, inbreeding, or sex, but is changed after freezing.