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.     

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.     

Dietary lipid sources affect cold tolerance of Nile tilapia (Oreochromis niloticus)

This study was carried out to evaluate the effects of dietary lipid sources on growth performance, fatty acids composition and cold tolerance of Nile tilapia (Oreochromis niloticus) fingerlings (7.00 ± 0.50 g/fish). The fish were fed four isonitrogenous (28% crude protein), isocaloric (500 kcal/100 g) diets containing four lipid sources; fish oil (FO), corn oil (CO), coconut oil (COCO) or fish oil/ corn oil mixture (1:1 ratio) (oil mix). The diets were offered to the fish at a daily rate of 3% of their body weights (BW), twice a day for two months. After the feeding trial, the fish were exposed to decreasing water temperature from 25 °C until the appearance of death symptoms. The results revealed that FO-based diets (FO and oil mix) produced the best growth rates and feed efficiency, followed by corn oil diet, while COCO resulted in the lowest performance. Fish fed on CO and oil mix showed higher body unsaturated fatty acids (UFA) and lower lethal temperature than those fed on FO- or COCO-based diets. These results indicate that cold shock can modify the lipid metabolism in Nile tilapia by lowering total body saturated fatty acids and raising n-6 and n-3 UFA. This finding suggests that the inclusion of high levels of plant oils in Nile tilapia feeds can enhance their cold tolerance.     

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.     

Sex reversal in Nile tilapia (Oreochromis niloticus) using a nonsteroidal aromatase inhibitor

Several studies have demonstrated that steroid hormones can influence sex differentiation in nonmammalian vertebrates and it has been hypothesized that male and female sex differentiation are driven by androgen and estrogen hormones, respectively. Estrogen biosynthesis is mediated by the steroidogenic enzyme cytochrome P450 aromatase, which converts androgens to estrogens. In the present study we examined the efficacy of a potent nonsteroidal aromatase inhibitor incorporated into the food, on sex reversal of Nile tilapia (Oreochromis niloticus) larvae. Nile tilapia larvae were divided in seven groups, which were fed with diets containing different amounts of the aromatase inhibitor Fadrozole (0, 50, 75 and 100 mg/kg) during 15 and 30 days, starting 9 days after hatching. Independent of the period, the proportion of males was significantly higher in the treated groups. Treatment with the highest doses (75 and 100 mg/kg) for 30 days produced 100% males. Histological examination revealed no differences in gonadal tissues between control males and treated fish. Furthermore, one intersex fish was identified in the group treated with 50 mg Fadrozole/kg for 30 days. This study reports that a 100% Nile tilapia male population can be obtained by suppressing aromatase activity and suggests that besides steroid hormones, nonsteroidal compounds, such as aromatase inhibitors, have potential for production of monosex population in tilapia. J. Exp. Zool. 290:177-181, 2001.     

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.     

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.     

The IMTA-cultivated Chlorophyta Ulva spp. as a sustainable ingredient in Nile tilapia (Oreochromis niloticus) diets

Increasing levels of a mixture of Ulva spp. produced in an integrated multi-trophic aquaculture (IMTA) system were evaluated in Nile tilapia juveniles for partial replacement of dietary fish meal. A control diet (CTRL) was compared with three experimental diets containing 10 % (U10), 15 % (U15), and 20 % (U20) of Ulva spp. meal. Triplicate groups of fish (13 g initial body weight) were fed each diet for 63 days at 26 °C. Nutrient apparent digestibility coefficients and nitrogen retention efficiency did not vary significantly among diets. By the end of the trial, all groups of fish more than tripled their initial body weight. Specific growth rate and final body weight of U10 diet were similar to CTRL and significantly higher than U15 and U20 diets. Increasing Ulva dietary incorporation levels significantly increased feed conversion ratio (FCR), from 1.0 (CTRL) to 1.4 (U20). Fish fed with U10 diet had the highest protein efficiency ratio and nitrogen retention efficiency allowing this fish to growth and reach a final body weight similar to the CTRL group. Protein content was highest in fish fed with the CTRL diet, whereas the highest lipid content was observed in fish fed with U20 diet. The results show that the incorporation of IMTA-produced Ulva meal in Nile tilapia diets is possible up to 10 % without compromising growth performance, protein utilization, and protein retention of juveniles. The high capacity of Nile tilapia to digest all experimental diets suggests that Ulva meal is a practical partial replacement for fish meal in Nile tilapia diets.     

Immunotoxicity and hepatic function evaluation in Nile tilapia (Oreochromis niloticus) exposed to diazinon

The LC(50) of the organophosphorus pesticides (OPs) diazinon to Nile tilapia (Oreochromis niloticus) was determined, thereafter, hepatic activity, phagocytic index, percentages of active cells, relative spleen weight, total IgM concentration and lymphoproliferation rates were compared between diazinon exposed groups (LC(50) and (1/2)LC(50)) and non-exposed control group. Experimental data show that diazinon is highly toxic for juvenile Nile tilapia (LC(50)=7.830 ppm) and presents immunotoxic properties which affect both the innate and cellular adaptive immune responses of this fish, as revealed by the fact that splenocyte proliferation and phagocytic indices were significantly decreased after acute exposure to the pesticide. However, the hepatic biochemical parameters and the total circulating IgM concentrations were not affected in this experimental model.     

Interactions between Nile tilapia (Oreochromis niloticus) and cladocerans in ponds (Khartoum,Sudan)

Juvenile Nile tilapia (Oreochromis niloticus) are omnivorous, and the question asked in this study is how they affect on their environment? Do they mainly act as predators on the cladoceran zooplankton or do they compete with the cladocerans for phytoplankton? This problem was studied in three ponds with and three ponds without small tilapia (3–5 cm). The fish growth rate, the succession of plankton species and the changes in abiotic conditions, were monitored over a period of 67 days. The fish biomass was kept low and the mean was approximately constant (12.6 g m^?2) during the experiment. Phosphate was added to avoid phytoplankton nutrient limitation. Although the diet of Nile tilapia contained both phytoplankton and zooplankton, the fish affected the ecosystem in a similar way as zooplanktivorous fish. The fish ponds got more phytoplankton due to increase of Chlorophyta. Effects on the other phytoplankton groups Euglenophyta, Bacillariophyta, Cryptophyta and Cyanophyta could not be registered. The ponds without fish had higher densities of Daphnia lumholtzi and D. barbata. The other Cladocerans seemed less influenced by fish presence. The relative fish growth rate was most positively correlated with the density of Daphnia lumholtzi, Diaphanosmoa excisum and Bosmina longirostris. Tilapia seemes to have two feeding modes: (1) preying on large zooplankton and (2) unselective filtration of small planktonic organisms such as phytoplankton. In our experiment the first feeding mode affected the ecosystem more than the second.     

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.     

Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae

A study was conducted to determine the role of specific antibodies in immunity to Streptococcus agalactiae. Adult Nile tilapia (Oreochromis niloticus) were injected i.p. with tryptic soy broth as control or with S. agalactiae vaccine. Ninety days later, fish were challenged with 1.5x10(4)CFUS. agalactiae fish(-1). Blood was drawn from all fish 90d after vaccination and 25d after challenge, and the acquired serum was injected i.p. in fingerling Nile tilapia. These passively immunized fish were subsequently challenged 72h later with 1.5x10(4)CFUS. agalactiae fish(-1), and significantly less (P<0.0001) mortalities were noted among fish administered serum containing specific anti-S. agalactiae antibodies (0.0-10.0% mortalities) than in control groups (63.3-72.7% mortalities). Heat-inactivation of serum produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P<0.9455) or vaccinated-challenged fish (P<0.0781). Pre-challenge serum samples indicate that the passively immunized fish had significantly increased (P<0.0001) specific antibody levels over control fish. A highly significant (r(2)=0.5892; P<0.0001) correlation between increased pre-challenge specific serum antibody OD levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results of this study indicate that specific anti-S. agalactiae antibodies play a primary role in immunity to S. agalactiae in fish.     

Protein and energy needs for maintenance and growth of Nile tilapia (Oreochromis niloticus)

A trial was undertaken with juvenile Nile tilapia fed graded levels of dietary protein (0–35% DP) over 7 weeks (28°C). Measurements of nitrogen and energy utilization were made using data on ADC, comparative carcass analyses, nitrogen excretion and oxygen uptake. Data indicate that the daily protein intake for maximum N gain was c. 12 g/kg/d and the maintenance protein requirements were about of 2g/kg/d. The optimal DP/DE ratio was found to be 18mg/kJ. Differences were observed in the data on endogenous nitrogen and energy utilization depending on the dietary treatments as well as on the criteria used.     

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.     

Daily rhythms after vaccination on specific and non-specific responses in Nile tilapia (Oreochromis niloticus)

ABSTRACT

We evaluated the daily changes in immunological and hematological factors in tilapia (Oreochromis niloticus) after an immunization period with a subsequent challenge. Experiments were divided into two phases: Phase 1 (immunization): 144 fish were distributed into two groups with 72 fish in six tanks. One group (T1) was immunized, comprising six vaccination time points (ZT schedule = ZT2 h, ZT6 h, ZT10 h, ZT14 h, ZT18 h, ZT22 h). The same schedule was applied to the other group, but with saline solution (non-vaccinated: T2). Both groups remained in the laboratory for 30 days (considered the immunization period). Phase 2 (challenge): on day 30, both vaccinated and non-vaccinated groups were challenged with Streptococcus agalactiae (2.0 × 10⁷ CFU mL^?1) following the same ZT schedule to stimulate the immune response without leading to widespread infection and mortality. On day 45, blood and head kidney samples were collected during the same ZT schedule. The variations in time of the following parameters within each group were evaluated: hematology, peroxidase activity, IgM, tnf-α3, tgf-β1, il-1β and il-12 gene expression. No significant mortality was observed for the groups or the ZT schedule (p > 0.05). Daily rhythms with diurnal acrophases were found in T2 for il12, tnf-α3 and tgf-β1 expression gene, while the acrophases of the peroxidase level, hematocrit and thrombocytes were at nighttime (p < 0.05). In contrast, most of the parameters in the vaccinated tilapia showed no daily rhythms (p > 0.05), except IgM. For all the parameters, the interaction effect between time and treatment (vaccinated and non-vaccinated groups) depended on ZT. Our results reveal that the humoral and non-specific immune system displayed a circadian rhythm based on the light-dark cycle, which could be affected by the vaccination procedure in tilapia.     

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.     

Nonadditive expression of lipid metabolism pathway-related genes in intestine of hybrids of Nile tilapia females (Oreochromis niloticus) and blue tilapia males (Oreochromis aureus)

Nonadditive expression contributes to heterosis in hybrids. In this study, the expression profiles of twelve lipid metabolism pathway-related genes were investigated in the intestine of Nile tilapia (Oreochromis niloticus) ♀?×?blue tilapia (Oreochromis aureus) ♂ hybrid. The expression of genes from the hybrid were assigned to nonadditive and additive expression pattern groups and compared with expression patterns from Nile tilapia and blue tilapia. In the intestine of the hybrid, apoA4B was expressed at intermediate levels, but apoB and MTP were assigned to ELD-B and ELD-N categories, respectively. The LPL and LRP1 showed transgressive up-regulation in the hybrid, but LDLR was assigned to the ELD-B category. For fatty acid uptake related genes, only FABP11a was categorized as nonadditive expression with transgressive up-regulation, while CD36 and FABP3 were categorized as additive expression in the intestine of the hybrid. Two genes in triacylglycerol metabolism, namely, FAS and DGAT2, showed transgressive up-regulation in the hybrid. Most of the genes analyzed in the present study showed nonadditive expression (8 in 12), and five genes showed transgressive up-regulation. These results indicated that the stimulation of lipid metabolism in the hybrid compared to that of its parents. The hyperactive expression of these genes in the hybrid may be associated with the growth and lipid usage vigor.

     

Effect of different stress factors on some physiological parameters of Nile tilapia (Oreochromis niloticus)

This study was conducted to determine the effect of different stress factors on some physiological measurements of Nile tilapia (Oreochromis niloticus).A total number of 160 Nile tilapia, the body weight ranging between 100 and 120 g, were exposed to three stress factors of hypoxia, overcrowding and starvation for different periods 24, 72 and 144 h. The results of cortisol level were 134.15, 144.27, 154.12 ng/ml and 140.18 ng/ml for control, hypoxia, overcrowding and starvation, respectively, while after 144 h did not show significant difference among treatments compared with control group. In contrast, the values of T₃ and T₄ observed reduction with significant difference that T₃ ranged between the highest value 122.12 ng/ml for control group to lowest value of starvation group 94.35, 93.81 and 88.46 ng/ml after 24, 72 and 144 h. Also, similar trend of results observed in T₄ and blood glucose among treatments. And the enzymatic activity of lactate dehydrogenise (LDH) increased in hypoxic group, while a significant reduction appeared in overcrowding and starved fish compared to control group. The pyruvate kinase (PK) activity decreased in hypoxic group but increased in other group.