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.     

Muscle growth in Nile tilapia (Oreochromis niloticus): histochemical,ultrastructural and morphometric study

Muscle growth in Nile tilapia (Oreochromis niloticus) was studied focusing on histochemical, ultrastructural, and morphometric characteristics of muscle fibers. Based on body length (cm), we studied four groups: G1=1.36+/-0.09, G2=3.38+/-0.44, G3=8.90+/-1.47, and G4=28.30+/-3.29 (mean+/-S.D.). All groups showed intense reaction to NADH-TR in subdermal fibers and weak or no reaction in deep layer fibers. In G3 and G4, an intermediate layer was also observed with fibers presenting weak reaction; in G4, groups of fibers with intense reaction were observed in the subdermal region. The myosin ATPase (m-ATPase) activities were acid-stable and alkali-labile in subdermal fibers; most deep layer fibers were alkali-stable and acid-labile. Intermediate fibers were acid-labile and alkali-stable. Two fiber populations were observed near deep muscle layer: one large presenting weak acid- and alkali-stable and the other small alkali-stable.During growth, muscle fiber hypertrophy was more evident in intermediate and white fibers for G3 and G4. However, in these groups, the presence of fiber diameters < or =21 microm suggested that there is still substantial fiber recruitment, confirmed by ultrastructural study, but hypertrophy is the main mechanism contributing to increase in muscular mass.     

Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus

The present study revealed a relationship between the kinetic change of apoptosis and the inflammatory response during experimental intraperitoneal infection with Edwardsiella tarda as a septicemic model. The morphological changes of apoptotic cells including cellular shrinkage, condensed nuclear chromatin, nuclear fragmentation and membrane blebbing were detected by light and transmission electron microscopy. TUNEL and agarose gel electrophoresis confirmed the fragmentation of DNA in the apoptotic cells. Apoptosis was highly detected in lymphoid organs prior to the inflammatory process and gradually decreased after an extensive inflammatory response. Apoptosis in thymus and spleen was extensive and an in vitro study revealed that lymphocytes were the major cell population which underwent apoptosis. The result suggests that E. tarda-induced systemic immunosuppression via lymphocyte apoptosis as determined by suppression of the systemic inflammatory response during an initial step of generalized septicemia.     

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.     

The blood-testis barrier and its breakdown following immunization to testis material in the Nile tilapia,Oreochromis niloticus

Summary The blood-testis barrier and its changes following immunization to testis material, were investigated by light- and electron microscopy in a teleost fish, the Nile tilapia Oreochromis niloticus, using horseradish peroxidase and bovine serum albumin as tracers. In the normal testis, histochemistry using horseradish peroxidase revealed that a barrier composed of junctional complexes connecting adjacent Sertoli cells existed around the central lumina of the seminal lobules, and also around the germ-cell cysts containing spermatids at the middle or late phase of chromatin condensation. By contrast, bovine serum albumin was prevented from passing through the basement membrane and could not penetrate any of the spermatogenetic cysts, indicating that the basement membrane may be an ion-selective barrier. In tilapia immunized with allogeneic testis homogenate emulsified in Freund's complete adjuvant, bovine serum albumin could penetrate the spermatogenetic cysts, and horseradish peroxidase was able to pass through the intercellular spaces between Sertoli cells to the region nearer the seminal lobule lumen, due to the junctional complexes becoming loosened. The results suggest that the blood-testis barrier, both junctional complexes and the basement membrane, are broken down during immune responses.     

A place preference test in the fish Nile tilapia

The Nile tilapia fish (Oreochromis niloticus) has a high potential to be used as a model in neuroscience studies. In the present study, the preference of the Nile tilapia between a gravel-enriched (GEE), a shelter-enriched (SEE) or a non-enriched (NEE) environment was determined, for developing a place preference model. Nile tilapia had an initial preference for GEE, but after 1 day of observation, the fish stabilized their frequency of visits among compartments. Hence, any stimulus motivating tilapia increase in compartment visiting indicates a positively reinforcing effect. This feature is very useful for the development of new behavioural paradigms for fish in tests using environmental discrimination, such as the conditioning place preference test.     

Effects of the organophosphate insecticide,monocrotophos, on acetylcholinesterase activity in the nile tilapia fish (Oreochromis niloticus) brain

The neurotoxic effects of monocrotophos on the brain of the nile tilapia fish (Oreochromis niloticus) were examined, using a static bioassay under laboratory conditions. By probit analysis the 96 h LC₅₀ value of monocrotophos was 4.9 mg/l. After 96 h exposure to acute levels of monocrotophos, the brain acetylcholinesterase (AChE) activity decreased progressively as the concentration of monocrotophos increased. In addition, four weeks following transfer to toxicant-free water after exposure to 1 mg monocrotophos, nile tilapia fish brain regained 95% of control AChE activity. The results indicate that inhibition of AChE activity in fish exposed to monocrotophos may serve as an indicator of hazard due to application of this chemical in the natural environment.Special issue dedicated to Dr. Robert Balazs.     

The breeding season of tilapia, Oreochromis niloticus L. in Lake Awassa (Ethiopian rift valley)

The breeding season of Oreochromis niloticus in Lake Awassa was studied from fish caught monthly from December 1987 to November 1988 in relation to a number of environmental factors. Fish at various stages of gonad development and spawning were caught in almost all months, but breeding fish were more frequent twice a year: main peak occurred during January–March and a secondary one occurred during July–September. Gonadosomatic index (GSI) values peaked twice in the study year, a major peak being during January–March and a less pronounced one during July–September. Thus, it was concluded that O. niloticus in Lake Awassa breeds intensively in the former and less intensively during the latter period, and that some breeding occurs at any time of the year. Intensive breeding activity during January–March appeared associated with increase in solar radiation and sunshine hours d^–1 whereas that during July–September appeared associated with heavy rainfall. However, intensive breeding in both periods coincided with increase in phytoplankton biomass. Previously, phytoplankton biomass in this lake is shown to increase following increase in nutrients due to mixing- and rainfall-associated changes in the hydrography and hydrology of the lake. Thus, although further studies are required, increase in phytoplankton may be one of the environmental cues to stimulate spawning in O. niloticus in Lake Awassa whereas other factors such as sunshine and rainfall may have indirect roles through their effects on the hydrology and hydrography of the lake ultimately resulting in changes in phytoplankton biomass.     

Molecular cloning of DAX1 and SHP cDNAs and their expression patterns in the Nile tilapia,Oreochromis niloticus

Piscine DAX1 and SHP cDNAs with an open reading frame encoding 296 and 258 amino acid residues, respectively, as well as SHP partial gene fragment, were cloned from Nile tilapia. Phylogenetic analyses of DAX1s, SHPs, and homologous EST fragments indicate that DAX1 and SHP are conserved in gene structure and are present throughout vertebrates. A single band of approximately 1.4kb for DAX1 and of approximately 1.2kb for SHP was detected in the Northern blot analysis. Tissue distribution analysis by RT-PCR showed that fish DAX1 and SHP mRNAs are widely expressed in adult tissues, with the most abundant expression in gonads and liver, respectively. DAX1 and SHP were also detected in gonads of both sexes at 5-90 days after hatching (dah). However, the expression of DAX1 is weak at 5 and 10dah and then significantly up-regulated between 10 and 15dah, whereas the expression of SHP is moderate and consistent during the ontogeny.     

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.     

Clock-controlled endogenous melatonin rhythms in Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus)

The purpose of this work was to investigate the circadian melatonin system in two tropical teleost species characterized by different behavioral habits, Nile tilapia (diurnal) and African catfish (nocturnal). To do so, fish were subjected to either a control photoperiod (12L:12D), continuous light (LL) or darkness (DD), or a 6L:6D photoperiod. Under 12L:12D, plasma melatonin levels were typically low during the photophase and high during the scotophase in both species. Interestingly, in both species, melatonin levels significantly decreased prior to the onset of light, which in catfish reached similar basal levels to those during the day, demonstrating that melatonin production can anticipate photic changes probably through circadian clocks. Further evidence for the existence of such pacemaker activity was obtained when fish were exposed to DD, as a strong circadian melatonin rhythm was maintained. Such an endogenous rhythm was sustained for at least 18 days in Nile tilapia. A similar rhythm was shown in catfish, although DD was only tested for four days. Under LL, the results confirmed the inhibitory effect of light on melatonin synthesis already reported in other species. Finally, when acclimatized to a short photo-cycle (6L:6D), no endogenous melatonin rhythm was observed in tilapia under DD, with melatonin levels remaining high. This could suggest that the circadian clocks cannot entrain to such a short photocycle. Additional research is clearly needed to further characterize the circadian axis in teleost species, identify and localize the circadian clocks, and better understand the environmental entrainment of fish physiology.     

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.     

外来养殖鱼类的入侵风险评估及防控对策——以尼罗罗非鱼为例

[目的]筛选合适的指标建立一套外来养殖鱼类的生物入侵风险评估体系,并对外来养殖鱼类的生物入侵防控提出对策建议。[方法]通过文献资料的收集和整理,对外来养殖鱼类中典型入侵物种的入侵过程、影响危害和入侵生物学特性进行分析和归纳,从适应能力、繁殖能力、扩散能力3方面指示其入侵性;从对生物的影响和对环境的影响2方面指示其生态影响;从自然因素和人为因素2方面指示环境可入侵性,以上述3方面为框架进行评估体系构建。[结果]筛选20个指标构建了外来养殖鱼类的生物入侵风险评估体系,并举例说明该评估体系的应用。从法规政策、科学研究、治理技术和公众参与等4个方面针对性地提出外来养殖鱼类生物入侵风险防控对策。[结论]防范和治理入侵生物是一个系统工程。对于外来养殖鱼类的管理,既不能只考虑经济效益而置生态风险于不顾,也不能片面放大外来养殖良种的入侵风险。科学管控的关键在于完善制度建设、加强风险评估、发展防治手段、促进公众参与,使外来鱼类养殖业在严格受控的前提下发挥其经济效益,将其潜在的生态危害效应降到最低。     

Evaluation of the influence of housefly maggot meal (magmeal) diets on catalase, glutathione S-transferase and glycogen concentration in the liver of Oreochromis niloticus fingerling

Influence of housefly maggot meal (magmeal) diets on the activities of catalase (CAT), glutathione S-transferase (GST) and glycogen concentration in liver of Tilapia Oreochromis niloticus fingerling was evaluated. Triplicate groups of fifteen fish (initial average weight 2.0+/-0.1 g) were fed eight weeks with seven test diets (in average 36% crude protein, dry matter) formulated by replacing fish meal with magmeal. Percentage body weight gain (591-724.46%), food conversion ratio (1.05-1.22) and standard growth rate (3.45-3.76) in all feeding groups were not significantly different (P<0.05). No significant difference (P<0.05) was observed in liver glycogen reserve (175.27-236.88 micromol g(-1)) among the fish groups. Hepatic catalase activity also did not differ significantly. However, elevated glutathione S-transferases activities were observed when fish received higher dietary magmeal concentration. This might have been temporary with no real physiological implication when appraised by the growth responses. These results indicate that magmeal was well utilized by the fish and its incorporation into tilapia diets seems to have no oxidative stress generating effect on fish metabolism and may not be containing any compound that stimulates the generation of reactive oxygen species. Magmeal can effectively be used as an alternative protein source in tilapia fingerling production.     

Time-place learning in food-restricted Nile tilapia

Time-place learning based on food association was investigated in eight food-restricted Nile tilapias. Each fish was individually housed for 10 days in an experimental tank for adjustments to laboratory conditions, and fed daily in excess. Feeding was then interrupted for 17 days. Training was then started, based on a food-restricted regime in a tank divided into three interconnected compartments. Daily food was offered in one compartment (left or right side) of the tank in the morning and on the opposite side in the afternoon, for a continuous 30-day period. Frequency of choices on the right side was measured on days 10, 20 and 30 (during these test days, fish were not fed). Following this 30-day conditioning period, the Nile tilapias were able to switch sides at the correct period of the day to get food, suggesting that food restriction facilitates time-place learning discrimination.     

Ultrastructural analysis of the developing follicle during early vitellogenesis in tilapia,Oreochromis niloticus,with special reference to the steroid-producing cells

The development and distribution of steroid producing cells (SPCs) in the ovary of tilapia have been studied by light and electron microscopy. At 40–50 d after hatching, these cells are seen only in the vicinity of blood vessels; there are no SPCs in the interstitial region, nor in the thecal layer enclosing young oocytes at the peri-nucleolus stage. By 70–80 d after hatching, the number of SPCs in the area near blood vessels has increased, and the capillaries have spread among the developing peri-nucleolar stage oocytes, and into the ovarian tunica. Clusters of SPCs have also migrated into the interstitial region and into the tunica along with these capillaries. In the ovary 100 d after hatching, some SPCs can be found in the thecal layer enclosing vitellogenic oocytes. Moreover, masses of SPCs can now be observed infiltrating the thecal layer of the oocyte. Serum testosterone (T) and estradiol-17 (E2) levels at 40–70 d after hatching, are low (T, 0.75–1.10 ng/ml, E2, 0.36–1.08 ng/ml), but at 100 d, plasma E2, but not T, is elevated (T, 1.95 ng/ml, E2, 4.65 ng/ml). These results suggest that SPCs appearing in the vicinity of blood vessels move into the interstitial region between oocytes, and finally enclose the oocytes at an early vitellogenic stage. It is interesting to note that the enclosure of oocytes by SPCs coincides with significant increases in E2 production.     

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.     

Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.     

The influence of social status on the rate of growth, eye color pattern and insulin-like growth factor-I gene expression in Nile tilapia, Oreochromis niloticus

Many aspects of teleost physiology are subject to regulation by social interactions. To evaluate the relationship of social status with growth, eye color pattern and hepatic Insulin-like Growth Factor-I (IGF-I) mRNA expression, 30 Oreochromis niloticus were isolated for 10 days and were used in a social pair study. Results revealed that growth of both dominant (except 1 day after social interaction) and subordinate individuals was suppressed, but growth suppression was greater in the subordinates. The dominant fish completely inhibited the feeding of the subordinate individuals during and 1 day after they were introduced into the aquaria together. After that, a pattern of highly aggressive attacks by dominant fish only partially inhibited feeding by the subordinates. Differential alterations in growth rate between dominants and subordinates were attributed more to behavioral changes (i.e., feeding) as transduced by physiological regulators (i.e., IGF-I level and possibly serotonin and/or neuropeptide Y) but may also be due to changes in metabolism. The fish's relative position in the social hierarchy consistently influenced the levels of IGF-I mRNA in the liver and the eye color pattern. Lower social status depressed hepatic IGF-I levels while dominant status stimulated hepatic IGF-I production, possibly in response to inhibition of somatostatin release in the hypothalamus, leading to greater secretion of pituitary growth hormone (GH). A significant positive association was detected between the IGF-I mRNA expression of the dominant fish and the level of aggression (number of attacks) during the encounter. Social status also influenced the eye color pattern of the fish. During aggressive interactions, most of the fish (22 out of 24) displayed decreased eye darkening. At the later part of the encounter, all subsequent subordinates displayed eye-darkening patterns which acted as a social signal announcing social submission. After the encounter dominant fish had paler eye color pattern than subordinates.