Genetic and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus)

Identification of the sex-determining genes of the Nile tilapia (Oreochromis niloticus) has important implications for commercial aquaculture. We previously identified an XX/XY sex-determining locus in this species within a 10-cM interval between markers GM201 and UNH995 on linkage group one (LG1). In order to refine this region, we developed new AFLP markers using bulked segregant analysis of the mapping families. We identified three AFLP markers that showed a sex-specific pattern of segregation. All three mapped near, but just outside, the previously identified sex-determining region on LG1. Hybridization of BAC clones containing these markers to chromosome spreads confirmed that the XX/XY sex-determining locus is on one of the small chromosomes in O. niloticus.     

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.     

遗传改良"新吉富" (NEW GIFT)尼罗罗非鱼RAPD-SCAR标记开发及其在品系鉴别中的应用

“新吉富”(NEW GIFT)尼罗罗非鱼是以1994年引进的 “吉富”(GIFT)品系尼罗罗非鱼为基础群体,经过14年9代系统选育后获得的新品种,该品种已被国家审定为良种,已在全国推广,迫切需要有效的分子遗传标记来鉴别该新品种并实施科学管理。该研究对良种群体开展了RAPD分析,并进一步将特异性的RAPD标记转化成了稳定的SCAR标记。 通过对“新吉富”群体的RAPD分析,找到了两个品种特异性的RAPD条带（S₃₀₄^{624 bp}和S₃₆^{568 bp}）。对品种特异性的RAPD条带进行了胶回收、克隆和序列测定。根据测序结果设计了两对位点特异性引物来扩增这两个品种特异性条带（简称SCAR标记Ⅰ和Ⅱ）,检测这两个SCAR标记在“新吉富”、“吉富”以及国内外7个养殖品系中的出现频率。SCAR标记Ⅰ（553 bp）在“新吉富”群体中的出现频率为85.7%,而在其基础群体（“吉富”）中的出现频率仅为16.7%;SCAR标记Ⅱ（558 bp）在“新吉富”群体中的出现频率达到91.4%,但在其它7个养殖品系中的出现频率仅在0%～70%之间。为验证这两个标记的可靠性,检测了这两个标记在一个埃及罗非鱼野生群体中的出现频率,发现SCAR标记Ⅰ和SCAR标记Ⅱ的出现频率分别为10%和70%,远低于在“新吉富”群体中的出现频率。这两个SCAR标记在“新吉富”良种群体中的高出现频率,预示它们可能与生长性能相关QTL位点间存在连锁关系,显示SCAR标记技术对选育世代间的纵向追溯、以及选育良种群体与其它养殖品系间的横向鉴别有较好应用前景。     

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.     

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.     

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.     

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.     

The introduced Oreochromis niloticus is spreading on the Kaufe floodplain,Zambia

The Nile tilapia, Oreochromis niloticus, escaped from a fish farm in Mazabuka, probably in 1987, and was found in September 1992 on a 75 km stretch of the river, mainly downstream from the site of release. In most areas, except near Mazabuka, O. niloticus was relatively uncommon and so far does not seem to have hybridized with the local tilapias.     

Otolith age determination for adult tilapia, Oreochromis niloticus L. from Lake Awassa (Ethiopian Rift Valley) by interpreting biannuli and differentiating biannual recruitment

Age of mature Oreochromis niloticus in Lake Awassa, Ethiopia, was estimated by analysing optical macrozones (translucent and opaque) in sagittal otoliths from fish sampled over a 12-month period. Seasonal record on the type of macrozone at the edge of otoliths suggested that two translucent macrozones associated with biannuli were formed each year; one during January and February and another during June and July. Formation of translucent macrozones coincided with minimum water temperature, spawning associated loss in condition and presumably with reduction in the quantity and quality of the food consumed by the fish. Relative marginal increment analysis showed that biannulus formation may be completed in March and in August each year. A concurrent study has confirmed a biannual recruitment in O. niloticus in Lake Awassa, from which mid-February and mid-August were taken as median hatch-dates. A procedure to assign otolith age is described which considers median hatch-dates and uses the number of biannuli in otoliths without discriminating between fish from the two recruitment cohorts. We also show a procedure to discriminate between the two cohorts by using the number of biannuli, conditions on the edge of the otolith and time of capture to assign age for the two recruitment cohorts separately.     

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.     

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.     

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。     

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.     

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.     

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.     

Ecological interactions between Nile tilapia (Oreochromis niloticus, L.) and the phytoplanktonic community of the Furnas Reservoir (Brazil)

1. Exotic invasive species modify natural food webs in a way frequently hard to predict. In several aquatic environments in Brazil the introduction of Oreochromis niloticus (tilapia) was followed by changes in water quality. Yet, because of its rapid and easy growth, this fish has been used in many aquaculture programmes around the country. 2. To measure the effects of tilapia on the phytoplankton community and on water conditions of a large tropical reservoir in south‐eastern Brazil (Furnas Reservoir), we performed two in situ experiments using three controls (no fish) and three tilapia enclosures (high fish density). Abiotic and biotic parameters were measured at 4 day intervals for 28 days. 3. Fish presence increased nitrogen (N) and phosphorus (P) availability (ammonium 260 and 70% mean increase – first and second experiment; and total phosphorus 540 and 270% mean increase) via excretion. Nutrient recycling by fish can thus be significant in the nutrient dynamics of the reservoir. The higher chlorophyll a concentration in the experimental fish tanks (86 and 34 μg L^?1, first and second experiment, respectively) was the result of a positive bottom‐up effect on the phytoplankton community (approximately 2 μg L^?1 in the reservoir and control tank). 4. Because tilapia feed selectively on large algae (mainly cyanobacteria and diatoms), several small‐sized or mucilaginous colonial chlorophyceans proliferated at the end of the experiments. Thus, the trophic cascade revealed strong influences on algal composition as well as on biomass. 5. Tilapia can contribute to the eutrophication of a waterbody by both top‐down and bottom‐up forces. In particular, by supplying considerable amount of nutrients it promotes the increase of fast growing algae. Tilapia must be used cautiously in aquaculture to avoid unexpected environmental degradation.     

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.     

Seasonal abundance,breeding and sex-structure of populations of Tilapia nilotica (L.) and Labeo niloticus (F.) in the Jebel Aulia Dam area of the White Nile

Seasonal abundance, maturation and sex-structure were studied in populations of T. nilotica and L. niloticus in the Jebel Aulia reservoir and in the White Nile below the dam. L. niloticus showed a single annual breeding cycle from mid-July to December and was most abundant in October in the lake, and in early November in the river. T. nilotica showed two distinct annual seasons of abundance: summer (April to June) and autumn (November to October). Spawning activity was highest in summer (April to June) and autumn (late July to September). The sex-ratio varied inversely with age (size); males were predominant in populations of older fish, perhaps because of their greater endurance to adverse conditions as evidenced by their better survival in hypoxic water.     

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.