Stability in aromatase immunoreactivity of steroid-producing cells during early development of XX gonads of the Nile tilapia, Oreochromis niloticus: an organ culture study

The organ culture system is a useful tool to study the effects of various factors on the development of undifferentiated gonads. In this study, we first established an organ culture system for gonads of all genetic male and female Nile tilapia at 5-122 days after hatching (dah). This short-term (3 days) organ culture system was then used to examine the stability of the immunoreactivity of aromatase (the enzyme which converts androgen to estrogen, thus playing a crucial role in ovarian differentiation) in steroid-producing cells (SPCs). Immunohistochemical analyses revealed that aromatase-positive cells could be initially detected in the vicinity of blood vessels in the XX gonads at 7 dah. These SPCs completely lost their immunoreactivity after 3 days in culture, indicating the instability of SPCs during early ovarian differentiation. In contrast, the immunoreactivity of the SPCs was maintained to some extent even after 3 days in culture, if the gonads were from 15-23 dah. In XX gonads collected at 122 dah, there were two major populations of SPCs: one in the vicinity of the blood vessel and the other near the oocyte. The aromatase immunoreactivity was maintained in SPCs located around the oocytes, but not in those in the vicinity of the blood vessel, after 3 days in culture. These results suggest that the SPCs originate from the cells in the vicinity of the blood vessels prior to the initial ovarian differentiation in tilapia and that the degree of differentiation of SPCs is dependent on their location in the ovary.     

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus)

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10⁹ cells mL⁻¹ and from 18′04″ to 27′32″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 μm s⁻¹ for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype.     

Nitrite-induced methemoglobinemia in Nile tilapia, Oreochromis niloticus

Exposure of Nile tilapia, Oreochromis niloticus (mean weight, 55.72 ± 4.30 g), to two sublethal NO₂–N concentrations was studied for 24 and 48 h in a static test. In nitrite exposure tests, the percentages of methemoglobin, external nitrite, plasma nitrite, hemoglobin and hematocrit were assessed. Nitrite exposure in the range of 0.50 and 1.38 mg l⁻¹ NO₂–N caused an increase in methemoglobin levels; however, methemoglobin percentages ranging from 16% to 42% represented a mild methemoglobinemia. Levels of methemoglobin were unrelated to environmental and plasmatic nitrite concentrations. The nitrite concentration in the blood did not seem to be linked to time of exposure. Nitrite exposure in Nile tilapia was associated with a marked reduction in hemoglobin and hematocrit.     

Orexin-B-like immunoreactivity localized in both luteinizing hormone- and thyroid-stimulating hormone-containing cells in the Nile tilapia (Oreochromis niloticus) pituitary

Immunohistochemical techniques were employed to examine orexin-like immunoreactivities in the pituitary of the Nile tilapia (Oreochromis niloticus). Rabbit anti-orexin-A serum and mouse anti-orexin-B monoclonal antibodies were used as primary antibodies. Orexin-B immunoreactive cells corresponded to luteinizing hormone (LH)- or thyroid-stimulating hormone (TSH)-containing cells, and all LH- and TSH-containing cells were immunoreactive for orexin-B. However, we found no orexin-A immunoreactive cells in the pituitary. In the Nile tilapia, an orexin-B-like substance may be secreted from LH- or TSH-containing cells and may regulate pituitary function, rather than the orexin-A-like substance in the pituitaries of Japanese seaperch and medaka.     

Metabolic and molecular responses in Nile tilapia,Oreochromis niloticus during short and prolonged hypoxia

The strictly aquatic breathing Nile tilapia, Oreochromis niloticus is an extremely hypoxia-tolerant fish. To augment our understanding of the effects of hypoxia on anaerobic glycolysis in the Nile tilapia, we studied the effect of short-term for 1 day (trial 1) and long-term for 30 days (trial 2) hypoxia on a selected glycolytic enzymes activity and mRNA expression in liver and white muscle. The hypoxic oxygen concentrations used in the two trials were 2, 1, and 0.5 mg O₂ L^?1 for comparison with a control normoxic group 8 mg O₂ L^?1. The activity of phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver and white muscle except liver LDH decreased in trial 1 and increased in trial 2. Assessments of mRNA levels in trial 1 revealed that PFK was downregulated and LDH was upregulated in liver and white muscle, while PK fluctuated between upregulation in liver and downregulation in white muscle. Meanwhile, PK and LDH were upregulated while PFK was similar to control values in both tissues in trial 2. Comet assay results demonstrated an increase in DNA damage that was directly proportional to increasing hypoxic concentrations. This damage was more pronounced in trial 1. This suggests that the Nile tilapia cope better with long-term hypoxic conditions, possibly as an adaptive response.     

Substratum choice for nesting in male Nile tilapia Oreochromis niloticus

Four substrata were offered to groups of adult Nile tilapia Oreochromis niloticus (one male and two females) simultaneously: pure sand, a mixture of sand and shells, stones and no substratum. The results showed that males chose to dig nests in a lighter and more homogeneous substratum.     

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.     

Identification and partial characterisation of a chitinase from Nile tilapia, Oreochromis niloticus

Measurement of chitinase activity in extracts from stomach, intestine, and serum of Nile tilapia with the artificial substrates 4-methylumbelliferil beta-D-N,N'-diacetylchitobioside and 4-methylumbelliferil beta-D-N,N'N"-triacetylchitotrioside (4MU[GlcNAc](2,3)) showed that an endochitinase was involved in the liberation of the fluorophore 4-methylumbelliferone (MU). Enzymes were isolated from tilapia serum by a combination of gel filtration, ion exchange, and reverse-phase chromatography. The molecular mass of the enzyme was estimated to be 75 kDa by SDS-PAGE, suggesting that the enzyme occurs as a monomer. The partially purified enzyme showed maximal activity at pH 7.0 when assayed with 4MU[GlcNAc](2) and lost its activity below pH 5.0 and above pH 8.0. The optimal pH of the purified enzyme toward the substrate 4MU[GlcNAc](3) was pH 9.0 and activity was lost below pH 8.0 and above pH 9.0. Our study has revealed the presence of a chitinolytic enzyme in the gastrointestinal tract and serum that may play a role in digestion and/or defense.     

A novel sex-determining QTL in Nile tilapia (Oreochromis niloticus)

Background

Fish species often exhibit significant sexual dimorphism for commercially important traits. Accordingly, the control of phenotypic sex, and in particular the production of monosex cultures, is of particular interest to the aquaculture industry. Sex determination in the widely farmed Nile tilapia (Oreochromis niloticus) is complex, involving genomic regions on at least three chromosomes (chromosomes 1, 3 and 23) and interacting in certain cases with elevated early rearing temperature as well. Thus, sex ratios may vary substantially from 50%.

Results

This study focused on mapping sex-determining quantitative trait loci (QTL) in families with skewed sex ratios. These included four families that showed an excess of males (male ratio varied between 64% and 93%) when reared at standard temperature (28°C) and a fifth family in which an excess of males (96%) was observed when fry were reared at 36°C for ten days from first feeding. All the samples used in the current study were genotyped for two single-nucleotide polymorphisms (rs397507167 and rs397507165) located in the expected major sex-determining region in linkage group 1 (LG 1). The only misassigned individuals were phenotypic males with the expected female genotype, suggesting that those offspring had undergone sex-reversal with respect to the major sex-determining locus. We mapped SNPs identified from double digest Restriction-site Associated DNA (ddRAD) sequencing in these five families. Three genetic maps were constructed consisting of 641, 175 and 1,155 SNPs from the three largest families. QTL analyses provided evidence for a novel genome-wide significant QTL in LG 20. Evidence was also found for another sex-determining QTL in the fifth family, in the proximal region of LG 1.

Conclusions

Overall, the results from this study suggest that these previously undetected QTLs are involved in sex determination in the Nile tilapia, causing sex reversal (masculinisation) with respect to the XX genotype at the major sex-determining locus in LG 1.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1383-x) contains supplementary material, which is available to authorized users.     

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.     

Comparative analysis of Hox paralog group 2 gene expression during Nile tilapia (Oreochromis niloticus) embryonic development

The hindbrain and pharyngeal arch-derived structures of vertebrates are determined, at least in part, by Hox paralog group 2 genes. In sarcopterygians, the Hoxa2 gene alone appears to specify structures derived from the second pharyngeal arch (PA2), while in zebrafish (Danio rerio), either of the two Hox PG2 genes, hoxa2b or hoxb2a, can specify PA2-derived structures. We previously reported three Hox PG2 genes in striped bass (Morone saxatilis), including hoxa2a, hoxa2b, and hoxb2a and observed that only HoxA cluster genes are expressed in PA2, indicative that they function alone or together to specify PA2. In this paper, we present the cloning and expression analysis of Nile tilapia (Oreochromis niloticus) Hox PG2 genes and show that all three genes are expressed in the hindbrain and in PA2. The expression of hoxb2a in PA2 was unexpected given the close phylogenetic relationship of Nile tilapia and striped bass, both of which are members of the order Perciformes. A reanalysis of striped bass hoxb2a expression demonstrated that it is expressed in PA2 with nearly the same temporal and spatial expression pattern as its Nile tilapia ortholog. Further, we determined that Nile tilapia and striped bass hoxa2a orthologs are expressed in PA2 well beyond the onset of chondrogenesis whereas neither hoxa2b nor hoxb2a expression persist until this stage, which, according to previous hypotheses, suggests that hoxa2a orthologs in these two species function alone as selector genes of PA2 identity.     

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.     

Effect of the hydrostatic pressure on otolith growth of early juveniles of Nile tilapia Oreochromis niloticus

Nile tilapia Oreochromis niloticus early juveniles were maintained for 2 weeks in a pressurized system under a controlled photoperiod, at constant salinity and temperature. Groups of fish were exposed to one of three absolute hydrostatic pressure (HP) regimes: (1) a constant normal atmospheric pressure (100 kPa), (2) a constant 40 m pressure (500 kPa) or (3) a semi-diurnal cyclic vertical migration (100-500 kPa). No significant differences were detected in otolith size and incremental periodicity among the three HP treatments, suggesting that HP does not affect otolith growth of early juveniles O. niloticus.     

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.     

尼罗罗非鱼早期发育形态及其在珠江水系的空间分布

【背景】罗非鱼作为联合国粮农组织（FAO）向全世界推广养殖的优良品种之一,有多个品系,其养殖范围已遍布85个国家和地区。1956年我国从越南引进莫桑比克罗非鱼,经过养殖及推广,2006年我国罗非鱼产量达到100万t。但是,该外来物种在给我国带来良好经济价值的同时,对土著种类及水环境造成了极大的影响。【方法】对近几年珠江水系渔获物的调查数据进行整理,并观察尼罗罗非鱼早期发育形态,统计尼罗罗非鱼苗对其他鱼苗的最大捕食量及捕食规格,以分析罗非鱼生物学特性及其在珠江水系的入侵现状。【结果】尼罗罗非鱼早期发育快,卵黄营养非常丰富,比珠江水系土著种类更有竞争性;罗非鱼苗呈现很强的攻击性与捕食性;尼罗罗非鱼已经扩散到珠江水系各主要河流,并在部分江段形成优势种群。【结论与意义】尼罗罗非鱼种群快速扩张,对土著种已构成严重威胁,有必要将其列为珠江水系高危入侵种。     

Identification of male-specific amh duplication,sexually differentially expressed genes and microRNAs at early embryonic development of Nile tilapia (Oreochromis niloticus)

Background

The probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development.

Results

Artificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY ‘supermales’ resulted in all-female and all-male embryos, respectively. RNA of pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20β-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10⁻⁹). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20β-hsd and amh. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exonVII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-β domain). amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns by a false discovery rate of p < 0.05. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20β-hsd, down-regulated in males.

Conclusions

This study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-β domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-774) contains supplementary material, which is available to authorized users.     

Taste preferences and feeding behaviour in the facultative herbivorous fish,Nile tilapia Oreochromis niloticus

Taste preferences in fishes are known mainly for carnivorous species, whereas herbivorous consumers were rarely used in such studies. The main goal of the present study was to evaluate the taste preferences in the herbivorous African cichlid fish, Nile tilapia Oreochromis niloticus. In laboratory settings, the palatability of widely used taste substances (four taste substances that are considered to be sweet, sour, bitter and salty for humans – sucrose, citric acid, calcium chloride and sodium chloride; 21 free L -amino acids; 12 sugars and artificial sweetener Na-saccharin; 0.1–0.0001 M) was evaluated. In each trial, a standard agar pellet flavoured with a substance was offered for fish individually. The consumption of pellet, the number of grasps and the retention time before the pellet was finally ingested or rejected were registered. Overall, 21 of 38 substances were palatable, whereas other substances did not shift consumption of pellets in relation to blank pellets. Pellets containing citric acid, L -cysteine, L -norvaline, L -isoleucine, L -valine, Na-saccharin and D -sorbitol were consumed in >85% of trials. Taste attractiveness of amino acids was highly species-specific and was not associated with the trophic category of the 19 species compared. Moreover, it did not correlate with dietary quantitative requirements of Nile tilapia (r_s = 0.27; P > 0.05). Palatability of sugars for O. niloticus and their sweetness for humans did not correlate as well (r_s = 0.21; P > 0.05); nonetheless, Na-saccharin has the most attractive taste for both O. niloticus and humans. The most palatable amino acids lost their effect if the concentration was lowered to 0.01 M for L -cysteine and 0.001 M for L -norvaline (lower than 242.3 μg and 23.4 μg per a pellet, respectively). Single pellet grasp was characteristic of O. niloticus feeding behaviour (>95% of trials), and this pattern may be related to the social lifestyle of this species. Fish spent 4–8 s on average for orosensory evaluation of pellet edibility. The retention time correlated with the palatability of substances and was significantly longer in trials that ended up with pellet swallowing. It is suggested that prolonged orosensory evaluation of food before swallowing provides a reliable and accurate sensory evaluation, which, in turn, can reduce the probability that inadequate food will be consumed.