Bone development in the jaw of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae)

East African cichlids have evolved feeding apparatus morphologies adapted to their diverse feeding behaviors. The evolution of the oral jaw morphologies is accomplished by the diversity of bone formation during development. To further understand this evolutionary process, we examined the skeletal elements of the jaw and their temporal and sequential emergence, categorized by developmental stages, using the Nile tilapia Oreochromis niloticus as a model cichlid. We found that chondrogenesis started in Stage 17. The deposition of osteoid for the dermal bones commenced in Stage 18. The uptake of calcium dramatically shifted from the surface of larvae to the gills in Stage 20. The bone mineralization of the skeleton began in Stage 25. These data provide important information regarding the sequential events of craniofacial development in East African cichlids and lay the groundwork for studying the molecular mechanisms underlying adaptation of jaw structure to feeding behavior.     

Development of the embryo, larva and early juvenile of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae). Developmental staging system

We described the developmental stages for the embryonic, larval and early juvenile periods of Nile tilapia Oreochromis niloticus to elucidate sequential events of craniofacial development. Craniofacial development of cichlids, especially differentiation and morphogenesis of the pharyngeal skeleton, progresses until about 30 days postfertilization (dpf). Because there is no comprehensive report describing the sequential processes of craniofacial development up to 30 dpf, we newly defined 32 stages using a numbered staging system. For embryonic development, we defined 18 stages (stages 1-18), which were grouped into seven periods named the zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatching periods. For larval development, we defined seven stages (stages 19-25), which were grouped into two periods, early larval and late larval. For juvenile development until 30 dpf, we defined seven stages (stages 26-32) in the early juvenile period. This developmental staging system for Nile tilapia O. niloticus will benefit researchers investigating skeletogenesis throughout tilapia ontogeny and will also facilitate comparative evolutionary developmental biology studies of haplochromine cichlids, which comprise the species flocks of Lakes Malawi and Victoria.     

Genetic characterization of four strains of Nile tilapia (Oreochromis niloticus L.) using microsatellite markers

Four domesticated strains of Nile tilapia (Oreochromis niloticus L.) were genetically characterized using 14 microsatellite markers and 64 animals per strain. Two strains, Chitralada (AIT) and International Development Research Centers (IDRC) were obtained from the AIT institute, Bangkok, Thailand. The GIFT strain (5th generation) came from NAGRI, Thailand, and the GÖTT strain was supplied by the University of Göttingen, Germany. The average numbers of alleles per marker were 5.0 (GÖTT), 5.4 (AIT), 5.6 (IDRC) and 7.5 (GIFT). Private alleles were found at all markers with the exception of two. No fixation of alleles was found at any marker. Population differentiation, F_ST, was 0.178 (great genetic differentiation) and confirmed grouping of the animals in strains. The expected level of heterozygosity ranged from 0.624 to 0.711, but the observed level of heterozygosity significantly deviated from the expected level in three strains. This was probably because of small population size. Moderate to great genetic differentiation was found between strains. A phylogenetic tree reflected the strains known histories. Application of the Weitzman approach showed that all strains have added value for the total genetic diversity and thus should be retained.     

Homogeneously sized groups increase aggressive interaction and affect social stress in Thai strain Nile tilapia (Oreochromis niloticus)

Social fish raised in farms are usually kept in groups of similar-sized individuals. However, social animals of similar size typically have similar fighting ability, which increases aggressive interaction for social rank establishment, as well as social stress. We compared Thai strain Nile tilapia fish, Oreochromis niloticus (L.), held under two treatments: (1) The Homogeneous one, with five adult male fish of similar size and (2) the Heterogeneous treatment with five adult males of different sizes. We recorded the frequency of aggressive interactions and checked social stability and stress levels (cortisol) after five days in the groups. Grouping similar sized Thai Nile tilapia increased the aggressive interactions and delayed rank stability with increased body injuries as a consequence. Homogeneous-sized individuals showed a similar level of stress while heterogeneous-sized individuals showed different stress levels with dominants being more stressed than subordinates. The data indicate that the practice of selecting fish of similar size in aquaculture management could reduce the welfare of social fish and that the effect is observed in different lineages.     

尼罗罗非鱼生长激素及其受体的cDNA克隆与mRNA表达的雌雄差异

尼罗罗非鱼(Oreochromis niloticus)雌雄鱼生长差异明显,为了探讨其原因,本文采用RT-PCR方法克隆了尼罗罗非鱼生长激素(Growthhormone,GH)及其受体(Growth hormone receptor,GHR)的cDNA序列,并应用半定量RT-PCR方法比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达差异。序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;GHR开放阅读框为1908bp,共编码635个氨基酸。以RT-PCR方法研究了GH、GHR在各组织的分布情况,结果表明:GH仅在垂体中检测到有表达,而GHR在所检测的18种组织中均有表达,其中以肝脏、肌肉、性腺、下丘脑、胸腺表达量较高。以半定量RT-PCR方法进一步比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达量,结果表明:雄鱼垂体GHmRNA和肝脏GHRmRNA的表达量均显著高于雌鱼,肌肉GHRmRNA的表达量则无显著差异,推测垂体GHmRNA和肝脏GHRmRNA表达的雌雄差异是尼罗罗非鱼雌雄生长差异的主要原因之一。     

尼罗罗非鱼Orexin前体基因的克隆、组织分布及其在摄食调控中的表达

该文采用RT-PCR和cDNA末端快速扩增技术(rapid-amplification of cDNA ends,RACE)的方法,从尼罗罗非鱼(Oreochromis niloticus)下丘脑总RNA中获得了尼罗罗非鱼Orexin前体基因的cDNA全长序列。该cDNA全长648bp,其中开放阅读框的长423bp,编码Orexin前体蛋白为140个氨基酸,包括37个氨基酸的信号肽、43个氨基酸的Orexin-A、28个氨基酸的Orexin-B和末尾32个氨基酸组成的功能不详的多肽。采用Real-time PCR技术对尼罗罗非鱼Orexin前体基因的组织表达模式以及在摄食前后、饥饿和再投喂状态下的表达量变化进行了研究。结果显示,在脑部和外周等18个组织中都检测到了Orexin前体基因的表达,其中在下丘脑中表达量最高;在摄食前后,尼罗罗非鱼Orexin前体基因的表达量显著低于在摄食状态中;饥饿2、4、6和8d后,Orexin前体基因在下丘脑中的表达量与正常投喂组相比均显著升高,饥饿4d再投喂后,表达量又恢复至正常水平。这些结果表明,Orexin在尼罗罗非鱼摄食中可能有着重要的调节作用。     

The protective influence of ascorbic acid against the genotoxicity of waterborne lead exposure in Nile tilapia Oreochromis niloticus (L.)

The present study investigated the effects of lead (Pb) and ascorbic acid (AA) on Nile tilapia Oreochromis niloticus using the micronucleus (MN) and nuclear abnormality (NA) tests for periods of 7, 14 and 21 days. The MN frequencies in the erythrocytes, gill, liver and fin cells were analysed comparatively to evaluate the sensitivity and suitability of these different cell types. The NA shapes in erythrocytes were scored into blebbed nuclei (BL), lobed nuclei (LB), notched nuclei (NT) and binuclei (BN). It was observed that fish showed significant sensitivity to the different treatments. In general, the highest value of both MN and NA cells were significantly increased in the Pb-treated group followed by the combination of the Pb and AA-treated group. On the other hand, the MN and NA frequencies in erythrocytes were the most sensitive to the treatment and could provide more valuable information than those in gill, liver and fin cells. The frequencies of each NA shape in erythrocytes of all treatments were observed in the following ranked order NT > LB > BN > BL. The results demonstrated the efficacy of AA in reducing genotoxicity in fish induced by Pb. They showed the sensitivity and suitability of MN and NA frequencies in erythrocytes as pollution biomarkers.     

Effects of exogenous melatonin on the reproductive activities of Nile tilapia,Oreochromis niloticus

Gonadal development and sexual maturation of fish are often related to photic conditions. We attempted to utilise the direct application of melatonin to elicit similar effects in the absence of photoperiod manipulation. We found no significant differences in somatic growth between melatonin-treated groups and controls, indicating that melatonin administration did not negatively affect the growth of the experimental fish. Treatment with low-dose melatonin (0.3 mg/kg BW) resulted in a decrease in spawning frequency, number of spawned eggs and gonadosomatic index in female tilapia. In male tilapia, low-dose melatonin was associated with a decrease in sperm count, spermatocrit and spermatozoa activity index, implying that exogenous melatonin might mimic the effects of shortened light photoperiod, which suppresses reproductive activity in this species. These results suggest the possibility that an optimised dose of melatonin treatment could be used to control the spawning behaviour of this species.     

Transgene activity following somatic transgenesis in Nile tilapia Oreochromis niloticus

A detailed study was made of the persistence and expression of a plasmid-enclosed reporter gene construct after intramuscular injection into the somatic muscle tissue of juvenile Nile tilapia Oreochromis niloticus and also the effect of injecting a potentially growth-promoting gene construct. The plasmid-enclosed DNA proved stable at the site of injection, lasting in some cases for up to 6 months, and was, at a very low frequency, detected in gonad tissue, indicating occasional substantial movement from the injected muscle site. It was observed that the reporter gene and regulatory sequences were also functional within the somatic cells. In a comparison of expression levels by direct somatic injection, the 1·6 kb tilapia β-actin regulatory sequence (tiβAP) resulted in c. three-fold higher β-galactosidase activity than the 4·7 kb carp β-actin regulatory sequence (cβAP) when spliced to the lacZ gene. The enhancer element near the end of first intron in the tiβAP, when co-injected with tiβAP/lacZ plasmid at a 3:1 ratio, drove significantly higher reporter activity in somatic cells than the tiβAP/lacZ sequence alone. The introduction of a growth-promoting construct, the Nile tilapia growth hormone gene driven by a tiβAP, yielded no detectable growth enhancement.     

Effect of melatonin on GnIH precursor gene expression in Nile tilapia,Oreochromis niloticus

Sexual maturation and gonadal development of fish is greatly influenced by photic information, an external environmental factor, and melatonin mediates this information to regulate gonadotropin (GTH) secretion and gonadal activation. The relationship between gonadotropin inhibiting hormone (GnIH) and melatonin in fish, however, has not been studied to date. Here, the GnIH expression pattern and daily change of melatonin levels were compared to each other in mature tilapia (body length 16.1 ± 0.2 cm, body weight 77.7 ± 3.43 g), and the effect of melatonin injection on GnIH gene expression was investigated. GnIH gene expression increased at night when the secretion of melatonin increased, whereas gene expression decreased during the day when melatonin secretion decreased. Injecting tilapia intraperitoneally with melatonin increased GnIH gene expression and decreased the expression of gonadotropin releasing hormone (GnRH) and GTH. Furthermore, the injection decreased the 11-KT concentration in male tilapia. These results indicate that melatonin is likely to suppress the hypothalamus-pituitary-gonad (HPG) axis via the action of GnIH in this species.     

Fine‐mapping of a locus on linkage group 23 for sex determination in Nile tilapia (Oreochromis niloticus)

Genetic markers in tilapia species associated with loci affecting sex determination (SD), sex‐specific mortality or both were mapped to linkage groups (LG) 1, 2, 3, 6 and 23. The objective of this study was to use these markers to fine‐map the locus with the greatest effect on SD in Oreochromis niloticus. Our parental stock, full‐sibs of Nile tilapia (Swansea origin), were divided into three groups: (i) untreated, (ii) feminized by diethylstilbestrol and (iii) masculinized by 17α‐methyltestosterone. We analysed the first group for association of microsatellite markers representing these five LGs. The strongest association with gender was found on LG23 for marker UNH898 (χ²; P = 8.6 × 10^?5). Allele 276 was found almost exclusively in males, and we hypothesized that this allele is a male‐associated allele (MAA). Sex‐reversed individuals were used for mating experiments with and without the segregating MAA. Mating of individuals lacking the MAA resulted in all‐female progeny. Mating of two heterozygotes for MAA gave rise to 81 males and 30 females. Analysis of association between gender and genotypes identified the MAA in 98.6% of males as opposed to 8.0% of females (χ²; P = 2.5 × 10^?18). Eight markers that flank UNH898 were genotyped to map the locus on LG23 within a confidence interval of 16–21 cM. Mating of homozygous individuals for MAA is underway for production of all‐male populations.     

Structure and enzymatic removal of the chorion of embryos of the Nile tilapia

Dechorionation or partial digestion of the egg chorion is necessary for introducing embryonic stem cells into blastulas for chimera production and for harvesting blastulas. Several methods to digest the Nile tilapia Oreochromis niloticus chorion were tried and it was found that the chorions of most clutches of eggs were digested in <3 h using hatching medium [produced by allowing embryos to hatch in Hanks balanced salt solution (HBSS) in an incubator], or 2 mg ml⁻¹ pronase P6911 in 10% Ca/Mg free HBSS. The chorion of Nile tilapia possesses multiple lamellae as found in most teleost species that have been studied. It was found to be thinner than that of medaka Oryzias latipes and thicker than that of zebrafish Danio rerio . During natural hatching the chorion was digested from the inner surface, and tail movements helped to break the remaining chorion; however, chorion digestion has to be complete for experimental dechorionation, because digestion starts at the external surface. The zona radiata externa remained intact after experimental digestion with hatching media but was disrupted by pronase. Embryos dechorionated at the cleavage or blastula stage only survived for 2 or 3 days, but some dechorionated at the gastrula stage or early segmentation stage developed until the natural hatching time. If the chorion was partially digested at the cleavage or blastula stage, some embryos survived to hatch.     

Circadian Rhythms of Locomotor Activity in the Nile Tilapia Oreochromis niloticus

Behavioral rhythms of the Nile tilapia were investigated to better characterize its circadian system. To do so, the locomotor activity patterns of both male and female tilapia reared under a 12:12 h light-dark (LD) cycle were studied, as well as in males the existence of endogenous rhythmicity under free-running conditions (DD and 45 min LD pulses). When exposed to an LD cycle, the daily pattern of activity differed between individuals: some fish were diurnal, some nocturnal, and a few displayed an arrhythmic pattern. This variability would be typical of the plastic circadian system of fish. Moreover, reproductive events clearly affected the behavioral rhythms of female tilapia, a mouth-brooder teleost species. Under DD, 50% (6 of 12) of male fish showed circadian rhythms with an average period (τ) of 24.1±0.2 h, whereas under the 45 min LD pulses, 58% (7 of 12) of the fish exhibited free-running activity rhythms with an average τ of 23.9±0.5 h. However, interestingly in this case, activity was always confined to the dark phase. Furthermore, when the LD cycle was reversed, a third of the fish showed gradual resynchronization to the new phase, taking 7–10 days to be completely re-entrained. Taken together, these results suggest the existence of an endogenous circadian oscillator that controls the expression of locomotor activity rhythms in the Nile tilapia, although its anatomical localization remains unknown.     

Identification of a sex-determining region in Nile tilapia (Oreochromis niloticus) using bulked segregant analysis

Sex determination in the Nile tilapia (Oreochromis niloticus) is thought to be an XX-XY (male heterogametic) system controlled by a major gene. We searched for DNA markers linked to this major locus using bulked segregant analysis. Ten microsatellite markers belonging to linkage group 8 were found to be linked to phenotypic sex. The putative Y-chromosome alleles correctly predict the sex of 95% of male and female individuals in two families. Our results suggest a major sex-determining locus within a few centimorgans of markers UNH995 and UNH104. A third family from the same population showed no evidence for linkage of this region with phenotypic sex, indicating that additional genetic and/or environmental factors regulate sex determination in some families. These markers have immediate utility for studying the strength of different Y chromosome alleles, and for identifying broodstock carrying one or more copies of the Y haplotype.     

我国新引进吉富品系尼罗罗非鱼群体的遗传多样性分析

选取罗非鱼(Oreochromis spp.)第二代遗传连锁图谱中的26个微卫星位点,对淡水渔业研究中心引进的、由60个家系组成的吉富品系尼罗罗非鱼(O.niloticus)群体进行遗传结构分析。结果显示,26个微卫星位点在吉富罗非鱼群体中共检测到124个等位基因,各位点的等位基因数为3～7个,平均4.8个。片段长度104～322 bp,平均杂合度观测值为0.622 1,平均杂合度期望值为0.642 3,平均多态信息含量(PIC)为0.633 4。所检测的26个位点中,有25个位点属于高度多态位点(PIC0.5),占所检测位点的96.15%;1个位点属于中度多态位点。结果表明,该吉富罗非鱼群体多态信息含量丰富,遗传多样性水平较高。因而该群体仍然具有较大的选育潜力,可以作为选育的基础群体开展进一步的选育工作。     

Effects of the pyriproxyfen and fenthion on the gonadal morphology of Nile tilapia (Oreochromis niloticus: Perciformes,Cichlidae)

Commercially available insecticides present acute toxicity to the health of fish and other aquatic organisms, which may impair the local aquaculture. This study evaluated the gonadal morphology of freshwater fish exposed to pyriproxyfen and fenthion. Forty-five juvenile male Nile tilapias (Oreochromis niloticus) were divided into control, pyriproxyfen-exposed (0.01 g/L), and fenthion-exposed (0.001 g/L) groups. They were evaluated in three moments (30, 60, and 90 days). The variables analyzed were the gonadosomatic index (GSI), weight to length ratio, seminiferous tubules morphometry (diameter and height), tissue damage, and immunohistochemical analysis for caspase-3, tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF). Pyriproxyfen and fenthion injured the seminiferous tubule tissue, and the damage progressed according to the exposure time. In addition, the GSI gradually reduced over time in all groups compared with the first moment (30 days), while caspase-3, TNF-α, and VEGF values increased only in the fenthion-exposed group. Therefore, pyriproxyfen and fenthion changed the gonadal morphology of male Oreochromis niloticus, which may affect their reproduction in the wild or captivity.     

Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus

A Foxl2 cDNA was cloned from the Nile tilapia ovary by RT-PCR and subsequent RACE. Alignment of known Foxl2 sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame and protein sequences, especially the forkhead domain and C-terminal region, while some homopolymeric runs of amino acids are found only in mammals but not in non-mammalian vertebrates. RT-PCR revealed that Foxl2 is expressed in the tilapia brain (B), pituitary (P), gill, and gonads (G), with the highest level of expression in the ovary, reflecting the involvement of Foxl2 in B-P-G axis. Northern blotting and in situ hybridization also revealed an evident sexual dimorphic expression pattern in the gonads. Foxl2 mRNA was mainly detected in the granulosa cells surrounding the oocytes. The ovarian expression of Foxl2 in tilapia begins early during the differentiation of the gonads and persists until adulthood, implying the involvement of Foxl2 in fish gonad differentiation and the maintenance of ovarian function.     

Duplication of neuropeptide Y and peptide YY in Nile tilapia Oreochromis niloticus and their roles in food intake regulation

In vertebrates, the neuropeptide Y (NPY) family peptides have been recognized as key players in food intake regulation. NPY centrally promotes feeding, while peptide YY (PYY) and pancreatic polypeptide (PP) mediate satiety. The teleost tetraploidization is well-known to generate duplicates of both NPY and PYY; however, the functional diversification between the duplicate genes, especially in the regulation of food intake, remains unknown. In this study, we identified the two duplicates of NPY and PYY in Nile tilapia (Oreochromis niloticus). Both NPYa and NPYb were primarily expressed in the central nervous system (CNS), but the mRNA levels of NPYb were markedly lower than those of NPYa. Hypothalamic mRNA expression of NPYa, but not NPYb, decreased after feeding and increased after 7-days of fasting. However, both NPYa and NPYb caused a significant increase in food intake after an intracranial injection of 50 ng/g body weight dose. PYYb, one of the duplicates of PYY, had an extremely high expression in the foregut and midgut, whereas another form of duplicate PYYa showed only moderate expression in the CNS. Both hypothalamic PYYa and foregut PYYb mRNA expression increased after feeding and decreased after 7-days of fasting. Furthermore, the intracranial injection of PYYb decreased food intake, but PYYa had no significant effect. Our results suggested that although the mature peptides of NPYa and NPYb can both stimulate food intake, NPYa is the main endogenous functional NPY for feeding regulation. A functional division has been identified in the duplicates of PYY, which deems PYYb as a gut-derived anorexigenic peptide and PYYa as a CNS-specific PYY in Nile tilapia.