Moussa Guèye1*, Justin Kantoussan2 and Mbaye Tine2
1IRD, route des hydrocarbures, BP 1386, 18524 Dakar, Sénégal
2UFR des Sciences Agronomiques de l’Aquaculture et des Technologies Alimentaires (UFR S2ATA), Université Gaston Berger (UGB), Route de Ngalléle BP 234, Saint-Louis, Sénégal
Received: 30 August, 2016; Accepted: 19 September, 2016; Published: 20 September, 2016
Moussa Guèye, IRD, route des hydrocarbures, BP 1386, 18524 Dakar, Sénégal, E-mail:
Guèye M, Kantoussan J, Tine M (2016) Common Garden Experiments Confirm the Impact of Salinity on Reproductive Traits that is Observed in Wild Populations of the Back-Chinned Tilapia Sarotherodon melanotheron. Int J Aquac Fish Sci 2(1): 031-037. DOI: 10.17352/2455-8400.000017
© 2016 Guèye M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Experiment; Impact; Reproduction; Salinity; Tilapia
The black-chinned tilapia Sarotherodon melanotheron is a very hardy species, particularly notable for its ability to tolerate a wide range of environmental salinities. The impact of environmental salinity on reproductive traits has been well documented in this species under natural conditions, but few studies have been experimentally conducted to prove such a relationship. This study assessed the effects of different salinities on the reproductive traits of S. melanotheron in experimental conditions. The number of spawning eggs as well as the mean number and frequency of incubation showed significant relationships with ambient salinity. The average number of incubations and spawning eggs were lower in hypersaline water (70 psu) compared the freshwater (0 psu) and seawater (35 psu). However the egg quality, as measured by the percentage of black eggs, was higher in hypersaline water. These variations in reproductive traits do not reflect genetic differences among individuals because fish used in common garden experiments were obtained by natural breeding from couples, the initial brood stock of which was from the same wild population. The results therefore confirm findings of previous studies in natural habitats, where variations in salinity are associated with differences in reproductive traits among populations. The lower number and frequent incubations recorded in hypersaline water could be an adaptive response to sustain the population dynamics despite the constraints imposed by extreme salinity.
The increasing demand for food resources due to growing human population has stimulated the intensification of tilapia farming in recent years, especially in Asian and South American countries such as Taiwan, Thailand, Philippines, Indonesia, Brazil and more recently in southern zones of United States, particularly in Florida and Texas . In Africa, tilapia were intensively cultured only in Egypt and Sudan until recently but, with the collapse of wild stocks, the farming of tilapines is growing rapidly in many countries . Tilapias have become the third most important species group of farmed fish in the world after carp and salmonids with a global production of approximately 3,200,000 metric tons in 2010 . The worldwide economic importance of this cichlid family is therefore growing rapidly, especially in countries where they represent an important source of dietary protein. This has led to increased research efforts for the improvement of aquaculture production, with focus on the identification of ideal environmental conditions for optimal growth and reproductive performance, best strain selection and sex control for fast growth and maximum productivity, and better feed conversion ratio [4-6]. For years, the intensive farming of tilapia for large-scale commercial production was almost exclusively limited to three species of the genus Oreochromis (O. niloticus, O. aureus and O. mosambicus) due to their best growth potential compared to the species of the genus Sarotherodon cultured in semi-commercial production systems essentially for local domestic consumption [7,8]. The growth rates obtained in the genus Sarotherodon are not impressive for commercial culture. However, the species has other potential such as fast growth, high adaptability and a wide dietary spectrum that promote its culture, which is now spreading to many countries of the world .
Although the growth potential of fish in aquaculture depends on the intrinsic growth potential of the species itself, environmental factors play also an important role. The black-chinned tilapia Sarotherodon melanotheron is known for its ability to tolerate a wide range of environmental factors including salinity. The species is one of the rare tilapia that can live in waters with very high salinities, up to 130 psu [10,11]. It is also probably the only tilapia that is able to reproduce in such high salinities although, at certain levels, salinity has marked influences on reproductive characteristics. Several comparative studies on wild populations have shown differences in reproductive traits such as age and size at maturity, and in fecundity, as a function of salinity, particularly in Senegalese and Gambian ecosystems where salinity shows considerable spatiotemporal variations [11,12].
These studies indicate that variations in salinity are the ultimate cause of these differences in reproductive traits, because they are the most dominant environmental factor, predominating over all other abiotic stressors. Nonetheless, this has not been rigorously tested. Many biotic/abiotic factors, such temperature, precipitation, competition, food availability and efficiency at foraging, could also directly or indirectly contribute to these differences. Furthermore, although unlikely, it is also conceivable that these differences also reflect genetic adaptation to local conditions. The species has strong population genetic structure, which is assumed to be linked to low larval dispersal ability due to their mouth-brooding reproductive behavior [13-15]. Genetic differentiation has been observed among populations even on a microgeographic scale in the Ebrié lagoon . It has also been found that samples collected at the same location at different times had more genetic differentiation than those collected at different locations of the lagoon, suggesting that the genetic structure of S. melanotheron populations is more dependent upon the degree of relatedness between individuals of the same generation than on geographical distance .
Common garden experiments are commonly employed to distinguish environmental from genetic influences on phenotypic variations among populations. These approaches allow separation of the genetic and environmental contributions to phenotypic variations and have been successfully used in a range of taxa including fishes [17-19]. When conducted with offspring from a single population with the same genetic background, common garden experiments can also be used to reveal which, among a group of environmental factors, is responsible for inter-individual variation in a given phenotypic trait in a single population . This is achieved by varying one environmental parameter while keeping the others constant.
In this study, we used common garden experiments to investigate the influence of ambient salinity on reproductive traits in S. melanotheron. The main aim was to confirm that differences in reproductive characteristics in wild populations in Senegalese and Gambian estuaries are related to spatiotemporal variations in salinity. Our approach also will give some indications on salinities for optimal reproductive and growth performance, which is crucial for farming of the species. Fish used in the experiments were from a single population maintained under laboratory conditions for several generations. Fish were exposed to different salinities (freshwater: 0 psu, seawater: 35 psu and hypersaline water: 70 psu) while other environmental factors and feeding conditions were held constant. More specifically, we assessed whether exposition to these salinity conditions induce differences in spawning number and frequency, the number, size and quality of spawned eggs, and the number of incubations as well as relation of these variables with body size.
Material and Methods
Experiments were performed on adults maintained at the GAMET (Groupe Aquaculture Méditerranéenne Et Tropicale) experimental facility in Montpellier, France. Fish were obtained by natural breeding and came from couples whose initial broodstock was collected from the Niayes region (Dakar, Senegal) and maintained at GAMET since 1998. Fish were reared for 16 weeks in six tanks of 430 liter each, two for each salinity condition (freshwater: 0 psu, seawater: 35 psu and hypersaline water: 70 psu). Young adults were reared in parallel in extra hypersaline water tanks to assess the impact of individual size on salinity tolerance and reproductive characteristics. Each tank was equipped with a thermostat to maintain a constant temperature of 27°C. The tanks functioned within a recirculated thermo regulated water system and each of them was equipped with a system of mechanical and biological filtration that maintained water quality high. Water was aerated vigorously to ensure optimal oxygen saturation. All tanks were initially filled with freshwater from a borehole and then salt was progressively added until the desired salinity (35 or 70 psu) was reached. The commercial salt mixture contained all the components of seawater and was initially dissolved in a bucket with water from the system before being diluted into the tank. For the hypersaline tanks, the salinity was increased by 2g of salt per liter of water every day until 70 psu. The rise in salinity was, however, stopped for two weeks after it reached 56 psu, due to high mortalities. The effective duration of the experimental follow-up phase was ten weeks and the phase of rise in salinity from 35 to 70 psu (considered an acclimation phase) took six weeks. Water was partially changed (about 1/3 of volume) at regular intervals to maintain quality high. Portions of PVC pipes were placed at the bottom of the tank to provide shelter and reduce aggressiveness between individuals in this territorial fish.
Tank stocking and physicochemical parameters
Before stocking the tanks, fish were anesthetized in a bath containing 0.3 ml/l phenoxy-2-ethanol, measured, weighed and marked individually with an electronic mark (PIT tag). Each tank was filled with ten fish comprised of five females and five males of equivalent size. Fish were fed with commercial processed fish feed containing 40% crude proteins and distributed six days a week for a fixed ration equivalent to 0.5-1% of the biomass. All individuals were monitored weekly to collect eggs in the male mouth brooders.
Physicochemical parameters (salinity, temperature, dissolved oxygen, ammonia and nitrite) were monitored regularly. Salinity was measured and adjusted once or twice per week using an optical refractometer. The temperature of the tanks was set at 27°C and maintained constant during the whole experimentation period. The concentration of dissolved oxygen was maintained around the saturation. These two parameters were monitored once or twice every week with an oximeter probe used for simultaneous measurements of water temperature and dissolved oxygen. The rate of nitrite and ammonium were controlled once or twice a week depending on the state of pollution in the tanks. Control of nitrate and nitrite was performed using a commercial kit used for simultaneous measurements of nitrite and ammonium concentrations.
Fish monitoring and control of incubation
The fish were monitored at the beginning of each week. During each test, the male incubators were identified by morphological and ethological criteria such as swelling of the lower part of the oral cavity, lack of aggressiveness, reduced feeding activity and mobility. Incubator males were captured with a net, and then forced to release the offspring in a water bucket by gently opening the mouth. The eggs were immediately recovered and stored in tubes containing water from the same tank.
Treatment of collected eggs
After collection, eggs were maintained alive in gently aerated water at their appropriate salinity. Developmental stages of the live eggs were immediately determined under an optical microscope, based on the Shaw and Aronson  scale in Tilapia macrocephala (synonym of the S. melanotheron). The diameter of the eggs was determined by image analysis with the Image J software. The eggs were then counted manually, and weighed to determine their total mass. A sample of 100 eggs was removed, weighed and the volume determined for the determination of egg density. Another sample of 100 eggs was weighed and dried at 105°C for 24 hours to determine water content. After drying, the sample was burnt at 550°C for 6 hours to determine ash content.
The condition factor (K), the most widely used morphometric index, provides information on the physiological state of the fish. Indeed, fish individuals that live in constrained environments generally have low body weight and size compared to those inhabiting favorable environments. The condition factor was compared between and within rearing environments at the beginning and the end of experience. Condition factor was calculated using the standard formula:
K = (P / LF3 ) * 105
where P is the total individual weight and the LF the fork length .
The number of incubations as a function of the number of females or males in the tanks was used as an index of reproductive rate in each rearing environment. This reproduction rate is indicated by the percentage of males incubators observed during successive samplings conducted in the course of the experimentation. This percentage also gave an idea of the number of females that were sexually active.
The U-test was used to compare the means of the salinity, temperature and O2 between 9h and 16h in each rearing environment. This was done to compare the variation of the daily experimental conditions between morning and afternoon during all experimental period. The variations of the salinity, temperature, O2, NH4 and NO2 between freshwater, seawater and hypersaline water were compared by using one-way ANOVA test. The same test was used to compare fish condition, oocyte weight and oocyte density between the three salinity conditions. To compare the number of incubations, number of spawning eggs, water and ash content in eggs between different water salinities, we used Kruskal-Wallis rank sum test. A linear regression was applied to test the relationship between the number of incubated eggs and the weight of male incubators. For all tests, a probability less than 5% was used as fiducial level of significance. All statistics were performed with the R statistical environment .
Table 1 shows that in each tank, the daily experimental conditions (between morning and afternoon) were comparable throughout the experimentation period. As previously mentioned, experimental tanks were initially filled with freshwater and then salt was progressively added until the desired salinity (35 or 70 psu) was reached. The differences in salinity between morning (9h) and afternoon (16h) were recorded during the phase of increase in salinity. The maximum daily salinity variation in SW and HW tanks during the increase in salinity were not significant (31.80±5.74 vs 33.06±0.51 for SW) and (35.92±12.31 vs 36.03±10.77 for HW) (Table 1). There were no significant changes in salinity, water temperature and dissolved oxygen between morning and evening (U-test, p>0.05). The comparison between rearing environments showed no significant differences in water temperature and dissolved oxygen (Table 2). The variation in ambient salinity between rearing environments was significant (Table 2). The levels of NH4 and NO2 were significantly different between rearing environments with higher values in seawater and hypersaline water tanks in comparison to freshwater tanks (ANOVA, p<0.05). Increased concentrations of nitrite were observed in seawater and hypersaline water tanks in the early phases of the experimentation, which were probably related to the fact that nitrifying bacterial flora was slower to develop in these environments.
Mortality rate in the rearing environments
The highest mortality rates were recorded in seawater (10%) and hypersaline water (15%) tanks while, in the freshwater tanks, mortality rate was 5%. The mortality rates were higher at the beginning of the experiment during the phase of rise in salinity from 15 to 35 psu for the seawater tanks and from 15 to 70 psu for hypersaline water tanks. Frequent mortalities were observed in the hypersaline thanks when the salinity reached 55 psu. The rise in salinity was therefore stopped for few days to allow fish to acclimate before continuing the experiment. In the three rearing environments, the highest mortality rates were recorded in females (40%) compared to males (20%). The aggressiveness of males, that can kill females, seemed to increase with salinity.
There were no significant differences in condition factor between individuals in the different tanks at the beginning of the experiment (ANOVA, p>0.05; Figure 1a) whereas at the end of the experiments, the average condition factor was higher in hypersaline water compared to the other rearing environments (freshwater and hypersaline water), with no significant difference between these latter conditions (ANOVA, p<0.05; Figure 1b). Condition factor decreased significantly with time in both freshwater and seawater tanks, but not in the hypersaline tanks (Table 3).