Molecular cloning and gene expression of the riboflavin-binding protein in the Nile tilapia, Oreochromis niloticus

Tilapia riboflavin-binding protein (RfBP) cDNA was isolated by library screening. Even though it shows only 31～35% similarity to the reported RfBPs, tilapia RfBP is conserved in amino acid residues that are known to be essential for its protein function. The exclusive expression of RfBP in the brain-pituitary-gonadal axis, as revealed by both RT-PCR and Northern blot, suggested a possible role for RfBP in fish reproduction.     

罗非鱼谷胱甘肽过氧化物酶1基因的克隆与分析

采用cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）技术,克隆了罗非鱼（Oreochromis niloticus）谷胱甘肽过氧化物酶1（glutathione pemxidasel,GPx1）基因完整的编码序列（complete coding sequence,CDS）。GPx1基因全长984bp,5’uTR56bp,CDS576bp,3’UTR352bp,PolyA20bp;第791—885位碱基（位于3’UTR）形成1个硒半胱氨酸插入序列（selenocysteine insertion sequence,SECIS）,协助174～176位密码子TGA（UGA）编码1个硒半胱氨酸（Sec）。GPx1包含191个氨基酸,分子量21．8kDa,等电点8．04,无信号肽和潜在的N．糖基化位点。蛋白结构分析表明该基因编码蛋白为非跨膜蛋白。序列比对显示,GPx1单体具有Sec、Trp、Gln和Asn构成的催化四联体。罗非鱼GPx1与其他脊椎动物GPx1相比较,核苷酸序列相似性为43．2％-58．2％,氨基酸序列相似性为58．1％～80．6％。进化分析显示,处在分类学上不同纲的脊椎动物GPx1分别占据了不同分支。利用Swiss-Model预测了罗非鱼GPx1的3D结构,序列分析显示,GPx1可以形成1个同源四聚体。     

Insulin-like growth factor-I cDNA cloning,gene expression and potential use as a growth rate indicator in Nile tilapia,Oreochromis niloticus

IGF-I is a mitogenic polypeptide that is an important regulator of growth in fish. The potential of IGF-I mRNA abundance as a rapid growth indicator in the Nile tilapia, Oreochromis niloticus, was evaluated. Hepatic IGF-I cDNA was isolated and partially cloned. The partial sequence having 539 bases encodes for the signal peptide, mature protein and a portion of the E domain. The deduced 68 amino acid sequence for mature IGF-I showed 84–90% and 77–79% sequence identity with fish and mammalian counterparts, respectively. The deduced amino acid sequence for domains B and A was most conserved (93–97%) relative to other fishes. A sensitive TaqMan real time qRT-PCR assay for O. niloticus was developed based on the mature IGF-I peptide for measures of hepatic IGF-I mRNA levels. Hepatic IGF-I mRNA levels were found to be significantly correlated with growth rate of fish reared under different feeding regimes and temperature conditions. Higher feed consumption and water temperature produced faster-growing fish and increased hepatic IGF-I mRNA expression. These findings suggest that hepatic-derived IGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species.     

Buprofezin toxication implicates health hazards in Nile tilapia (Oreochromis niloticus)

The objective of this study was to investigate the toxic effects of buprofezin insecticide on Nile tilapia (Oreochromis niloticus). The fish were exposed to buprofezin at 100 mg/L for 28 days. Compared to control, activity of serum transferases and levels of urea and creatinine showed significant increases. Oxidative stress was recorded manifested by elevated levels of malondialdehyde (MDA), reduced concentrations of reduced glutathione (GSH) and inhibition of activities of superoxide dismutase (SOD) and catalase (CAT) in liver and kidney. Examination of peripheral RBCs revealed elevated frequency of micronucleated cell. Interleukin 1 beta (IL‐1β) gene was upregulated in liver, muscle and brain, while that of cyclooxygenase 2 (COX‐2) gene increased in liver and muscle, but not in brain. Histopathological alterations were recorded in liver, kidneys, brain, gills, pancreas, spleen, intestine, muscle and ovaries. The immunohistochemical detection of caspase‐3 in the liver revealed no differences between treated and control groups; however, the expression of inducible nitric oxide synthase (iNOS) was demonstrated in hepatocytes and hepatopancreas in buprofezin‐treated group compared to control. It has been concluded that the tissue damage induced by buprofezin in Nile tilapia is mediated by oxidative stress and inflammatory response but not by apoptosis.     

低温胁迫对尼罗罗非鱼水通道蛋白(Aquaporin 1，AQP1)基因(AQP1)表达的影响

以经过连续多代抗寒选育获得的尼罗罗非鱼(Oreochromis niloticus)耐寒品系为实验材料, 克隆了尼罗罗非鱼水通道蛋白基因(AQP1)的cDNA序列, 并采用实时荧光定量PCR分析方法探讨了低温胁迫对罗非鱼AQP1基因表达水平的影响。序列分析结果显示, 尼罗罗非鱼AQP1 cDNA长度为1 098 bp, 包含36 bp的5′端非翻译区序列、783 bp开放阅读框(ORF)和279 bp的3′非翻译区序列, 编码261个氨基酸。氨基酸序列比对表明, 各物种间AQP1序列的同源性较高(96%~59%)。聚类分析表明, 尼罗罗非鱼首先与同属鲈形目丽鱼科的斑马拟丽鱼(Maylandia zebra)聚在一起, 再与其他硬骨鱼类聚为一类。实时荧光定量PCR 结果表明: 在水温从30℃逐渐降到10℃过程中, 肌肉和肝组织AQP1基因的表达量从20℃开始均逐渐下调。其中肌肉组织中, 与30℃时的表达量相比, 耐寒品系在20℃时表达量大幅度下调, 当水温为15℃时, 其表达量下调幅度为22.16倍。当水温降到10℃时, 其表达量下调了107.73倍。而对照组AQP1基因的表达量在15℃时, 下调幅度为5.38倍。当水温达到10℃时, 其表达量下调幅度为11.30倍。结果分析显示, 耐寒品系与对照组AQP1基因在低温条件下的表达量存在显著性差异(P<0.05)。而在肝组织中, 当水温降到15℃时, 耐寒品系和对照组的表达量下调幅度分别为3.38倍、1.42倍。水温降到10℃时, 其表达量上调幅度分别为18.85倍、9.01倍。结果分析表明, 低温条件下耐寒品系与对照组AQP1基因在肝组织中的表达差异不显著(P>0.05)。结果表明, AQP1表达对温度敏感, 耐寒品系较高的耐寒能力与其在低温条件下的肌肉组织大幅上调表达有关。由此可见, AQP1基因在罗非鱼低温适应过程中发挥重要作用, 是潜在的研究罗非鱼耐寒机制的候选基因之一, 本研究为进一步研究罗非鱼的耐寒分子机制和开展鱼类抗逆育种提供参考。

     

Spermatogenesis in teleost: insights from the Nile tilapia (Oreochromis niloticus) model

The morphometric study of spermatogenic cysts in sexually mature tilapias, during the evolution of spermatogenesis, showed a dramatic increase in both number of germ cells and cyst volume. However, the opposite trend was observed for germ cell size. Nevertheless, the number of Sertoli cells increased gradually up to leptotene/zygotene cysts, stabilizing thereafter. Based on the number of spermatids supported by each Sertoli cell and compared to mammals, Sertoli cell efficiency in tilapias is remarkably high. Sertoli cell proliferation was frequently observed, mainly in spermatogonial cysts, and probably is the major factor related to the testis growth and the increase in sperm production that normally occurs in adult tilapias. The combined duration of spermatocytes (5 days) and spermiogenic (5–6 days) phases of spermatogenesis in fish kept at 25 °C was 10–11 days. Mainly due to acceleration in meiosis, these two phases lasted a total of 6 days in tilapias kept at 30 °C, in the opposite way, at 20 °C spermatogenesis was arrested at pachytene spermatocytes. To our knowledge, this is the most comprehensive investigation performed up to date on testis morphometry and function in adult tilapias.     

Glycerol inclusion in the diet of Nile tilapia (Oreochromis niloticus) juveniles

The present study evaluated the effects of inclusion of glycerol in the diet of Nile tilapia juveniles on growth performance, biochemical changes in blood, and carcass composition. We used 300 Nile tilapia juveniles with an average initial weight of 29.15 ± 8.40 g and 11.55 ± 0.87 cm in length, distributed in 20 fiberglass tanks with a capacity of 250 L. The experiments followed a completely randomized design with five treatments and four replications during 79 days. The animals were fed diets containing four concentrations of glycerol (25 g kg^?1, 50 g kg^?1, 75 g kg^?1 and 100 g kg^?1) and a control diet without glycerol. HDL was the only biochemical parameter, that showed statistically different (P < 0.05) results; it was higher in the groups fed with 0 and 75 g kg^?1 glycerol compared to the other groups. No significant difference was observed in the results from the carcass composition of tilapia juvenile fed with the different glycerol levels, except for lipids (P < 0.05), which showed the highest values in fish fed with 50 g kg^?1 glycerol and the lowest in fish fed with 100 g kg^?1. Glycerol can be used in fish diets as an energy supplement without causing damage to growth performance or to the biochemical and carcass composition of Nile Tilapia juveniles.     

Sex reversal in Nile tilapia (Oreochromis niloticus Linnaeus) by androgen immersion

Two experiments were conducted to investigate the efficacy of three androgens applied through immersion treatments on the sex ratio of Nile tilapia (Oreochromis niloticus L.) fry. In experiment 1, 14 days post‐hatching (DPH) larvae were exposed to a single immersion treatment in 17α‐methyltestosterone (MT), 17α‐methyldihydrotestosterone (MDHT) or 17α‐ethynyltestosterone (ET) at 200, 600 and 1800 μg L^?1 over 4 h (130 larvae per treatment). In experiment 2, Nile tilapia larvae were exposed to the higher androgen concentration (1800 μg L^?1) applied as either a single immersion (14 DPH) or double immersion (10 and 14 DPH) over 4 h (125 larvae per treatment). Change in sex proportion within each experiment as well as between experiments was analysed by the chi‐square test. In experiment 1, MT, MDHT and ET were equally effective in significantly increasing the proportion of males when applied at 1800 μg L^?1 (86.0%, 90.0% and 86.7% respectively). At 200 μg L^?1 none of the androgens altered sex ratio. At 600 μg L^?1, only MDHT slightly, but significantly skewed the sex ratio towards males (73.0%). In experiment 2, a single immersion treatment at 14 DPH (1800 μg L^?1) significantly increased the proportion of males, but at this time the response was significantly hormone dependent (MDHT, 100.0%; MT, 91.6%; ET, 76.9%). When compared with a single immersion, two‐immersion treatments significantly increased the proportion of males in the MT‐treated group (from 91.6% to 98.3%), decreased the proportion of males in the MDHT group (from 100.0% to 93.4%) and had no significant effect the ET‐treated group (change from 76.9% to 82.5%). The overall comparison of the sex ratio among same treatments from different experiments (a single immersion in 1800 μg L^?1) was not significantly different.     

Alteration in expression of atrogenes and IGF-1 induced by fasting in Nile tilapia Oreochromis niloticus juveniles

The growth rate of farmed fish is an important factor regarding aquaculture success. An understanding of the cellular events that occur in skeletal muscle when fish undergo periods of fasting and refeeding provides information useful in developing alternative feeding strategies for improving muscle growth in commercially cultivated species. To evaluate the effect of 1–3 weeks of fasting and 10 weeks of refeeding in Nile tilapia juveniles, we analyzed the growth performance and changes in muscle cellularity and the expression of the following growth and muscle related genes: MyoD, myogenin, IGF-1, IGF-1 receptor, MuRF-1, atrogin-1 and myostatin. Reduced body mass was observed in all three groups of fasted fish during their time off feed, and 10 weeks of refeeding resulted in partial compensatory growth of body mass. No differences in the frequency of white muscle fiber diameters were observed between fasted and fed control fish treatments. However, changes in gene expression induced by fasting and refeeding were found. IGF-1 receptor, ubiquitin ligases MuRF1 and atrogin-1 expression increased during the 1–3 weeks of fasting, while IGF-1 levels dropped significantly (P < 0.001) compared to the control treatment. Furthermore, myogenin mRNA level in fish submitted to 3 weeks of fasting was higher in comparison to the control treatment (P < 0.05). Overall, our results showed that 1–3 weeks of fasting can induce muscle atrophy activation in Nile tilapia juveniles, and 10 weeks of refeeding is enough to induce only partial compensatory growth.     

Depletion of stored nutrients during fasting in Nile tilapia (Oreochromis niloticus) juveniles

We evaluated growth performance and metabolic responses in Nile tilapia (Oreochromic niloticus) juveniles (30.2 ± 0.9 g) subjected to 1 (F1), 2 (F2), or 3 weeks (F3) of fasting and then refed for 10 weeks (10WR) compared to controls (FC), which were fed for the full 13-week trial. Weight gain and specific growth rate (SGR) during fasting were lower in all treatments compared to the FC. However, during refeeding, feed intake/body mass and SGR increased in F1, F2, and F3, inducing partial compensatory growth. The hepatosomatic index (HSI), visceral fat index (VFI), liver glycogen (LG), and carcass lipid levels dropped in all fasted fish compared to FC (P < 0.05), showing a depletion of stored nutrients such as fat and LG. Along with LG, fat reserves were mobilized during fasting to maintain basal metabolism and survival, but these energy constituents returned to control levels at 10WR, at which time HSI was higher in all refed fish compared to FC. Additionally, the variables VFI, LG, and lipid in carcass increased in all refed fish, equaling those of FC at 10WR. The results showed that, in contrast with other protocols that used smaller tilapia juveniles, the feeding strategies utilized for Nile tilapia juveniles in this study (1 to 3 weeks of fasting and 10WR) were able to induce only partial compensatory growth. It can be concluded that in situations that require complete food restriction in juvenile Nile tilapia (30 g), an acceptable strategy is to limit the period of fasting to 1 week or less to minimize losses and to achieve partial compensatory growth.     

雄尼罗罗非鱼肝脏2种生长激素受体基因表达的发育性变化

选取同一批30日龄的尼罗罗非鱼(Oreochromis niloticus)鱼苗置于水泥池中自然饲养,分别于30、60、80、110、140日龄取样测定不同发育阶段雄鱼的特定生长率.用实时荧光定量PCR方法检测雄鱼肝脏中两种生长激素受体(GHR1和GHR2)的mRNA丰度.结果表明,30～110日龄,雄鱼特定生长率呈不断上升趋势,至80～110日龄达到最高,之后开始下降;雄鱼肝脏中2种GHR基因表达的发育性变化趋势不相同,30～110日龄,GHRi mRNA表达逐渐上升,110日龄达到最高,随后开始下降,其发育性变化与特定生长率显著正相关(R=0.96,P<0.01);GHR2 mRNA表达在60日龄达到最高.80日龄急剧下降,之后又缓慢上升,其发育性变化与特定生长率无相关性.提示尼罗罗非鱼生长激素可能主要通过与肝脏中GHR1受体的结合启动其促生长机制.肝脏中GHR1 mRNA表达具有显著的年龄依赖性,其表达对尼罗罗非鱼的生长有重要影响.[中国水产科学,2009,16(1):1-7]     

Genetic diversity and differentiation of Nile tilapia (Oreochromis niloticus) revealed by DNA microsatellites

To assess the genetic diversity of Nile tilapia Oreochromis niloticus, a total of 250 fish from five Egyptian populations were genotyped using six microsatellite markers. Heterozygosity and Wright's F‐statistics (F_IS, F_ST, and F_IT) were calculated to determine the genetic variation within and between these populations. Observed heterozygosities were in the range of 0.4 (Burullus) to 0.96 (Qena), with F_IS values ranging from 0.082 to 0.282. The mean F_ST showed that approximately 96.5% of the genetic variation was within‐population and 3.5% was among populations. Standard genetic distances were used to classify the five populations into two major groups. The deeper lotic river Nile populations of Assuit and Cairo formed one group and the shallow less lotic Delta lakes populations of Manzalla and Burullus formed the second group, with the upstream Nile Qena population being an outgroup. The findings from the current study help understanding of the broad‐scale population structuring of the Nile tilapia (O. niloticus) allowing the population groupings identified to act as potential sources of genetic variation. These populations could be included in future Marker‐Assisted‐Selection programs for economically desired production traits.     

Characterization and expression analysis of the transferrin gene in Nile tilapia (Oreochromis niloticus) and its upregulation in response to Streptococcus agalactiae infection

In this study, full-length tilapia transferrin (OnTF) isolated from liver cDNA of Nile tilapia (Oreochromis niloticus) was found to have an open reading frame of 2,091-bp encoding 696 amino acid residues. Two additional amino acids: Gly³⁶⁹ and Gly³⁷⁰ were observed compared with the reported Nile tilapia transferrin protein sequence. Pre-mature protein has a predicted molecular weight of 78.2 kDa, while mature protein is 73.28 kDa in size. Comparative sequence analysis with transferrin from other species revealed two major putative iron-binding domains designated as the N-lobe and the C-lobe in accordance with the transferrin protein characteristics. The predicted tertiary structure of tilapia transferrin confirmed the presence of iron and anion-binding sites on both lobes that are conserved among transferrins from other species. Quantitative real-time PCR analysis showed significantly higher expression of tilapia transferrin gene in liver than in other tissues (p < 0.05). Transferrin expression in tilapia experimentally infected with 10⁶ and 10⁸ colony-forming units mL^?1 of Streptococcus agalactiae was significantly upregulated at 24 and 12 h post-infection (hpi), respectively, and decreased afterward. Iron-deficiency in serum of bacterially infected fish was detected at 48 and 24 hpi, respectively. The expression pattern of the transferrin gene and the iron levels of infected tilapia in this study were consistent with the function of transferrin in innate immunity.     

Combined effects of temperature and salinity on yolk utilization in Nile tilapia (Oreochromis niloticus)

The combined effects of temperature and salinity on the yolk utilization of sac fry in Nile tilapia (Oreochromis niloticus) were investigated using central composite experimental design and response surface approach. Based on the preliminary trials, temperature was determined to range from 22 to 34°C, and salinity ranging from 2 ppt to 10 ppt. The utilization was mensurated in terms of yolk sac volume. Results showed that the linear effects of temperature and salinity on the yolk utilization was significant (P < 0.01); the quadratic effects of and the interaction between the two factors were significant (P < 0.05); temperature was more important than salinity in influencing the yolk utilization. The model equation of yolk sac volume towards temperature and salinity was established. From those high R² values, the model had excellent goodness of fit to experimental data and could be applied for predictive purpose. What with the production cost, it is suggested that the temperature/salinity combination, i.e. 28–30°C/4–6 ppt, be employed during the period of sac fry rearing, in which the yolk utilization was on average 98.6%.     

A new approach to fishery waste revalorization to enhance Nile tilapia (Oreochromis niloticus) digestion process

The main goal of this research was to analyse in vitro compatibility of Nile tilapia (Oreochromis niloticus) digestive proteinases and enzymes recovered from species comprising fishery waste as Pleoticus muelleri, Artemesia longinaris and Patagonotothen ramsayi. It evaluated the use of exogenous enzymes as feed supplements to increase digestion efficiency in tilapia fingerlings and juveniles (3.5 ± 0.11 g and 11.6 ± 1.5 g, respectively). We successfully have obtained proteinases from fishery waste as source of exogenous enzymes. P. muelleri and A. longinaris enzymes had more activity of acid and alkaline proteinases than P. ramsayi. SDS‐PAGE gels demonstrated that Nile tilapia digestive proteinases keep their activity when combined with each exogenous proteinase. Exogenous enzymes varied in their ability to enhance hydrolysis of different feed ingredients. P. muelleri by‐products are the best candidates to be employed as feed supplements for tilapia juveniles. Enzymes from this by‐product did not affect the activity and integrity of fish digestive enzymes, improved the hydrolysis of different protein sources (fish meal, squid meal, shrimp meal and soybean meal), and maintained its activity after being exposed to high temperatures and acid pHs. Our findings are applicable to other places where O. niloticus is raised utilizing local fishery waste, and also to different cultured species.     

Nile tilapia (Oreochromis niloticus) invasions disrupt the functional patterns of fish community in a large subtropical river in China

Although non‐native species can modify ecosystem function, their effects in southern China have not been investigated, despite their ecological importance. This study assessed how Nile tilapia Oreochromis niloticus (L.) invasions impact the functional patterns of fish communities in the large subtropical Pearl River by analysing the relationships between invasion and body size over a 9‐year period. The relative abundance of Nile tilapia increased significantly over time. In particular, the established Nile tilapia significantly undermined the relative densities and the body size of the native fishes. Importantly, this study provided empirical evidence that functional indices (such as body size) are more sensitive when evaluating the effects of non‐native species on a community than richness. Understanding processes such as those should be the basis of controlling alien fish species and fisheries management in the Pearl River.     

Total replacement of fish meal with animal protein sources in Nile tilapia,Oreochromis niloticus (L.), feeds

The effects of total replacement of dietary fish meal (FM) with animal protein sources on the growth, feed efficiency and profit indices of Nile tilapia, Oreochromis niloticus (L.), were investigated. Shrimp meal (SM), blood meal (BM), meat and bone meal (MBM), BM + MBM mix and poultry by-product meal (PBM) replaced FM in six isonitrogenous (30% crude protein), isocaloric (400 kcal GE 100 g^–1) diets. The diets were fed to O. niloticus fingerlings (12.5 g) to satiation twice a day for 150 days. The growth of fish fed SM, PBM and MBM was not significantly different from those fed the FM-based diet, while feed conversion and protein efficiency ratios were significantly retarded. Further reduction in fish performance was noticed when BM or BM + MBM replaced FM in the control diet. Cost–benefit analyses of the test diets indicated that these sources were economically superior to FM. The PBM-based diet produced higher carcass lipid than other diets. Fish fed SM, MBM and PBM diets had significantly higher ash contents (P < 0.05).