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.     

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.     

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.     

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.     

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.     

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.     

Growth and IGF-I response of juvenile Nile tilapia (Oreochromis niloticus) to changes in water temperature and dietary protein level

Water temperature and dietary protein level play an important role in influencing the growth and insulin-like growth factor I (IGF-I) in Nile tilapia juveniles. The combined effect of temperature (20–34 °C) and dietary protein level (25–50%) on the specific growth rate (SGR), feed efficiency (FE), serum IGF-I level and hepatic IGF-I mRNA level was examined under laboratory conditions by employing central composite design and response surface method. Results showed that the linear effects of temperature and dietary protein level on the SGR, FE, serum IGF-I and hepatic IGF-I mRNA level were significant (P<0.05); the quadratic effects of temperature and dietary protein level on the FE and serum IGF-I were significant (P<0.05). The interaction of temperature and dietary protein level on the FE, serum IGF-I and hepatic IGF-I mRNA level all proved significant (P<0.05). The optimal temperature/dietary protein level combination was determined, i.e., 29.9 °C/40.3%, at which the greatest SGR (2.748%/d) and FE (0.775) were simultaneously arrived. Both SGR and FE were linearly correlated with serum IGF-I or hepatic IGF-I mRNA level. These results suggested that optimum combination of temperature and dietary protein level would enhance tilapia growth efficiency and IGF-I would regulate growth and FE.     

Higher parasite richness, abundance and impact in native versus introduced cichlid fishes

Empirical studies suggest that most exotic species have fewer parasite species in their introduced range relative to their native range. However, it is less clear how, ecologically, the loss of parasite species translates into a measurable advantage for invaders relative to native species in the new community. We compared parasitism at three levels (species richness, abundance and impact) for a pair of native and introduced cichlid fishes which compete for resources in the Panama Canal watershed. The introduced Nile tilapia, Oreochromis niloticus, was infected by a single parasite species from its native range, but shared eight native parasite species with the native Vieja maculicauda. Despite acquiring new parasites in its introduced range, O. niloticus had both lower parasite species richness and lower parasite abundance compared with its native competitor. There was also a significant negative association between parasite load (abundance per individual fish) and host condition for the native fish, but no such association for the invader. The effects of parasites on the native fish varied across sites and types of parasites, suggesting that release from parasites may benefit the invader, but that the magnitude of release may depend upon interactions between the host, parasites and the environment.     

Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 μg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 ± 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T₁ were fed on basal diet (control). The fish of T₂ were given the basal diet, containing propolis-ethanolic-extract. The fish of T₃ were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 × 10⁷ cells ml⁻¹) at the end of the feeding period and kept for 15 more days.The best growth rate and feed conversion ratio were obtained with T_2. The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T₂ followed by T₃ when compared with the control group. The HCT-level and monocyte-counts were increased (T₂). No significant change, in the large lymphocytic-count was found among the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T₂ and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T₂. The RLP against A. hydrophila was high with T₂ and T₃.The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.     

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.     

Vocalization toward conspecifics in silver foxes (Vulpes vulpes) selected for tame or aggressive behavior toward humans

We examined the production of different vocalizations in three strains of silver fox (unselected, aggressive, and tame) attending three kinds of behavior (aggressive, affiliative, and neutral) in response to their same-strain conspecifics. This is a follow-up to previous experiments which demonstrated that in the presence of humans, tame foxes produced cackles and pants but never coughed or snorted, whilst aggressive foxes produced coughs and snorts but never cackled or panted. Thus, cackle/pant and cough/snort were indicative of the tame and aggressive fox strains respectively toward humans. Wild-type unselected foxes produced cough and snort toward humans similarly to aggressive foxes. Here, we found that vocal responses to conspecifics were similar in tame, aggressive and unselected fox strains. Both cackle/pant and cough/snort occurred in foxes of all strains. The difference in the use of cackle/pant and cough/snort among these strains toward humans and toward conspecifics suggest that silver foxes do not perceive humans as their conspecifics. We speculate that these vocalizations are produced in response to a triggering internal state, affiliative or aggressive, that is suppressed by default in these fox strains toward humans as a result of their strict selection for tame or aggressive behavior, whilst still remaining flexible toward conspecifics.     

Seasonal variation in seed bank composition and its interaction with nutrient enrichment in the Everglades wetlands

In the Florida Everglades, nutrient enrichment from agricultural outflow and the change in hydrology have collectively contributed to the expansion of cattails (Typha spp.). To assess the effectiveness of prescribed fire in controlling cattails and to predict vegetation dynamics after the fire, it is important to understand the seasonal variation of the soil seed bank and how the seed bank is affected by nutrient enrichment and fire. This paper investigates the effects of season, nutrient enrichment, and fire on soil seed bank species composition, richness, and density along a nutrient gradient in Water Conservation Area 2A (WCA 2A) of the Florida Everglades. Species richness was significantly affected by nutrient enrichment and season but not their interaction. Total seed density, however, was significantly affected by the interaction between nutrient enrichment and season. Yet, at species level, the relationship between seed density, nutrient enrichment and season varied. The highest seed density of cattail occurred in summer at highly enriched sites, but that of sawgrass occurred in fall regardless of enrichment; the seed density of water lily was very low regardless of season and nutrient enrichment, and the highest Amarathus seed density occurred at highly enriched sites year round. Moreover, germination timing differed greatly among species. While cattail seeds had a short incubation period and started to germinate 2–3 days after initiation of the germination assay, sawgrass seeds generally started to germinate 4 weeks later. Further, both the prescribed summer fire at the highly enriched site and the natural winter fire at the moderately enriched site reduced the seed density of cattail but not of sawgrass. Our results suggest that fire application for vegetation recovery in WCA 2A would benefit from explicitly considering seasonal dynamics of the seed bank.     

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.     

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.     

Androgen in free-living arctic ground squirrels: seasonal changes and influence of staged male-male aggressive encounters

We tested the responsiveness of plasma androgen in free-living male arctic ground squirrels (Spermophilus parryii) to challenges from conspecific males by staging aggressive encounters between males and then comparing androgen concentrations in manipulated and unmanipulated animals. The effect of staged encounters on androgen depended on time of year. In spring, androgen levels significantly increased after staged encounters, except during the peak of mating activity when levels were maximal in all reproductive males. In late summer, staged encounters had no significant effect on androgen levels. In spring but not summer, androgen was positively correlated to the relative intensity of encounters. These results suggest that circulating levels of androgen in male arctic ground squirrels result from a combination of seasonal and social factors. Responsiveness of androgen to encounters during spring but not late summer support the challenge hypothesis for this polygynous mammal.     

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.     

Local adaptation and enhanced virulence of Nosema granulosis artificially introduced into novel populations of its crustacean host, Gammarus duebeni

Local adaptation theory predicts that, on average, most parasite species should be locally adapted to their hosts (more suited to hosts from local than distant populations). Local adaptation has been studied for many horizontally transmitted parasites, however, vertically transmitted parasites have received little attention. Here we present the first study of local adaptation in an animal/parasite system where the parasite is vertically transmitted. We investigate local adaptation and patterns of virulence in a crustacean host infected with the vertically transmitted microsporidian Nosema granulosis. Nosema granulosis is vertically transmitted to successive generations of its crustacean host, Gammarus duebeni and infects up to 46% of adult females in natural populations. We investigate local adaptation using artificial horizontal infection of different host populations in the UK. Parasites were artificially inoculated from a donor population into recipient hosts from the sympatric population and into hosts from three allopatric populations in the UK. The parasite was successfully established in hosts from all populations regardless of location, infecting 45% of the recipients. Nosema granulosis was vertically (transovarially) transmitted to 39% of the offspring of artificially infected females. Parasite burden (intensity of infection) in developing embryos differed significantly between host populations and was an order of magnitude higher in the sympatric population, suggesting some degree of host population specificity with the parasite adapted to its local host population. In contrast with natural infections, artificial infection with the parasite resulted in substantial virulence, with reduced host fecundity (24%) and survival (44%) of infected hosts from all the populations regardless of location. We discuss our findings in relation to theories of local adaptation and parasite-host coevolution.     

Effect of water temperature,salinity, and their interaction on growth,plasma osmolality,and gill Na,K-ATPase activity in juvenile GIFT tilapia Oreochromis niloticus (L.)

We used a central composite rotatable experimental design and response surface methodology to evaluate the effects of temperature (18–37 °C), salinity (0–20‰), and their interaction on specific growth rate (SGR), feed efficiency (FE), plasma osmolality, and gill Na⁺, K⁺-ATPase activity in GIFT tilapia juveniles. The linear and quadratic effects of temperature and salinity on SGR, plasma osmolality, and gill Na⁺, K⁺-ATPase activity were statistically significant (P<0.05). The interactive effects of temperature and salinity on plasma osmolality were significant (P<0.05). In contrast, the interaction term was not significant for SGR, FE, and gill Na⁺, K⁺-ATPase activity ⁽P>0.05). The regression equations for SGR, FE, plasma osmolality, and gill Na⁺, K⁺-ATPase activity against the two factors of interest had coefficients of determination of 0.944, 0.984, 0.966, and 0.960, respectively (P<0.01). The optimal temperature/salinity combination was 28.9 °C/7.8‰ at which SGR (2.26% d¹) and FE (0.82) were highest. These values correspond to the optimal temperature/salinity combination (29.1 °C/7.5‰) and the lowest plasma osmolality (348.38 mOsmol kg⁻¹) and gill Na⁺, K⁺-ATPase activity (1.31 µmol Pi. h⁻¹ g⁻¹ protein), and resulted in an energy-saving effect on osmoregulation, which promoted growth.     

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.