Reviews in Aquaculture, 1–12
Nutrition, growth and resilience of tiger grouper (Epinephelus fuscoguttatus) 3 giant Grouper (Epinephelus lanceolatus) hybrid- a review Rossita Shapawi, Faihanna Ching Abdullah, Shigeharu Senoo and Saleem Mustafa Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia
Correspondence Saleem Mustafa, Borneo Marine Research Institute, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia. Email: [email protected]
Received 28 May 2018; accepted 3 September 2018.
Abstract The hybrid grouper discussed in this paper is a cross between female tiger grouper (Epinephelus fuscoguttatus) and male giant grouper (Epinephelus lanceolatus). Performed for the first time at this institute, the hybridization was motivated by the need to meet grouper demand. The hybrid has been a subject of thorough scientific investigations ever since it was produced. Qualities such as dietary needs, efficiency in growth and production and environmental resilience are of considerable aquaculture advantage, and account for interest in its commercial-scale farming in the wider Asia-Pacific region. This paper reviews scientific evidences on tolerance of hybrid grouper to environmental variability, nutrition, growth and genetic and other aspects. It makes serious attempt to summarize the pertinent data published on specific research questions to improve understanding of the diverse evidences, and to be able to identify gaps in knowledge. This has helped in articulating the current state of research and defining topics for future studies on the hybrid. From the analysis of published data it is obvious that hybrid has a higher production potential and resilience. Nevertheless, the hybrid is vulnerable to health problems linked to nutritional deficiencies and other factors. More comprehensive data on dietary requirements of the hybrid, especially larval stages, will help in formulating feeds that cater to metabolic requirements and improve the survival and biomass gains. Information on hybrid’s dietary flexibility can be used in developing feeds for grow-out stages comprising ingredients from sustainable sources. The hybrid could be a suitable candidate for adapting aquaculture to climate change. Key words: grouper, heterosis, hybridization, production, sustainability.
Introduction Groupers are among the most popular fish in aquaculture and a highly traded seafood commodity in the Asia-Pacific region (FAO, 2018). Intense fishing pressure and unsustainable methods of exploitation have depleted their populations and inflicted considerable damage to coral reefs that provide habitat to this high-value group of fish (Teh et al. 2005; Scales et al. 2007; Khatib 2015; Waheed et al. 2015). Regular visits to the landing centres and fish markets over the years revealed a decreasing proportion of large-sized fish in the catch, skewness in sex ratio in this protogynous fish, decline in catch per unit effort and heavy pressure on immature virgin class. All these observations suggest that much remains to be done in eradicating illegal, unreported © 2018 Wiley Publishing Asia Pty Ltd
and unregulated (IUU) fishing (Oakley et al. 1999; Pilcher & Cabanban 2001; Burke et al. 2012). Obviously, with this condition of grouper stocks, the pressures are beyond their reproductive and recruitment capacity, and threshold limits in the marine biodiversity hotpots in the coral reef ecosystems (Mustafa 2015). The International Union for Conservation of Nature has also raised the level of concern for these keystone species. It is realistic to presume that only aquaculture can meet the grouper demand and lessen the pressure on the overfished populations of not only this group of fish but also the marine biodiversity in general. Interest in grouper aquaculture is driven by declining supply from capture fishery at a time when there is an unprecedented increase in demand. While grouper farming has provided social and economic benefits to the coastal 1
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communities in Asia, the practices followed have raised concerns about their environmental impacts. The criticism is due to harvesting of wild-caught juveniles, dependence on prey fish as the main feed source, use of antibiotics to control infection and the effluent discharge. For coastal regions in tropical Asia the socio-economic contribution of grouper aquaculture is significant enough to deserve solutions to these problems. Shortage of high-quality seed is a major constraint to production of farmed grouper (El-Gamal 2001; Hassan et al. 2015). This appears to be linked to broodstock infertility, inbreeding depression, malnutrition, heavy larval mortality, stress and vulnerability to diseases. Despite considerable research and advances in hatchery technologies, adequate supply of quality fingerlings has remained an elusive goal. This is due to shortage of broodstock, late maturity, difficulty in closing its cycle, heavy larval mortality, bottleneck and founder effects, inbreeding and other related problems (Harvey et al. 2016). Worst sufferers are the small-scale farmers and entrepreneurs since the fish provides them income for livelihood. Borneo Marine Research Institute considered many options to address these problems and intensified research on environment-friendly and low-carbon aquaculture methods. While these methods received a great deal of interest but the groupers remained in increasing demand, mainly because of the growing local market, dietary preference of people and premium price of this popular seafood in the international market. In an attempt to boost the seed supply of the groupers, researchers of this Institute performed trials involving hybridization of the tiger grouper, Epinephelus fuscoguttatus (female) and giant grouper, Epinephelus lanceolatus (male) using native stocks. Success was achieved by Senoo (2006). This first-in-the-world hybridization of these species marked a turning point in grouper aquaculture in the region. The hybrid now supports the small-scale farmers as well as the industry, and is believably taking some pressure off the unsustainable exploitation of coral reef ecosystems. Before the successful production of tiger grouper x giant grouper hybrid (henceforth referred to as TGGG), attempts have been made on other species combinations. Worth mentioning in this connection is the work of Tseng and Poon (1983) who hybridized white-spotted green grouper (E. amblycephalus) and red grouper (E. akaara). The authors were able to trace the various developmental stages in fertilized egg until hatching and growth of the larvae. The hybrid so produced combined the qualities of both the parents. Subsequently, Glamuzina et al. (1999) succeeded in crossing the Mediterranean groupers, E. marginatus 9 E. aeneus, and James et al. (1999) in crossing E. fuscoguttatus with E. polyphekadion (camouflage grouper). 2
Hybridization of E. lanceolatus and E. coioides was performed by Kiriyakit et al. (2011) using cryopreserved sperm of the former species. Of the various extenders tested, the Marine Fish Ringer and Sodium Citrate resulted in high rate of fertilization. There was no significant difference between the performance of two cryoprotectants, namely, Dimethyl sulfoxide and Trehalose. Success of cryopreservation in maintaining the viability of giant grouper sperm for extended periods will help in making the much sought-after gamete of this species available for use by fish breeders who can mature females of other species under captivity in the hatcheries. Sutthinon et al. (2015) crossed E. lanceolatus and E. coioides and observed the tolerance of the hybrid to a range of salinities. TGGG has been a subject of intense investigations. There is a great deal of interest in understanding its unique features, especially those of aquaculture advantage (Table 1). Notable findings are elaborated in this paper. Growth One of the main considerations in selecting a fish for aquaculture is its growth. This helps in determining how fast a fish can attain harvestable size and yield commercial returns. Most of the studies have been based on measurement of biomass and effect of various culture conditions on growth rate. However, the mechanism of growth process in TGGG was examined in detail because achieving faster rate of growth was among the main motives for its production. While there is a genetic basis for higher growth rate of the hybrid grouper, but the somatic growth of the body can be modulated by many factors, including diet, and physical and chemical factors of the culture system. Therefore, the genetic as well as environmental factors influencing the phenotypic trait of growth were examined for better understanding the benefits from the culture of hybrid grouper. In this connection, studies carried out so far provide a good indication of their phenotypic plasticity (the ability to exhibit different phenotypes in response to environmental factors) in traits like growth. Stimulating growth by environmental factors enhances the genetic growth advantage which augurs well for aquaculture production. Some noteworthy publications reporting the effects of different diets and environmental factors on growth and general condition of the fish are reviewed here. De et al. (2016) studied the effect of diet and temperature on growth and gastric emptying rate of TGGG. They noticed that the temperature elevation between 26°C and 30°C positively affected the growth whereas increase to 34°C and decrease in the range 26–22°C adversely affected this process. Evidently, the optimum temperature range for growth of the hybrid is large enough to support its growth Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
Aquaculture performance of grouper hybrid
Table 1 Review of research on tiger grouper 9 giant grouper hybrid (TGGG)
Table 2 Growth performance of TGGG juveniles under different salinity treatments. Source: Othman et al. (2015)
1 2 3 4 5 6 7 8
9 10 11
12 13 14 15 16 17 18 19 20 21 22 23
Topic of research Method of hybridizing tiger grouper 9 giant grouper Egg and larval development Effect of temperature and salinity on survival and food intake Gonad development and maturation Effect of acidification of seawater Embryonic development and morphological changes Effect of temperature on gastric emptying Effect of salinity on growth, feed preference and plasma cortisol level Effect of diet composition on growth and feed utilization Optimum dietary protein level and protein-to-energy ratio Effect of dietary protein and lipid on growth and body composition Feeding habits and growth dynamics Growth performance and disease resistance Optimum temperature for growth Acceptability of soybean meal-based diets Effect of corn starch on growth and feed utilization Effect of salinity on hatching and embryonic development Molecular mechanisms of growth Dietary ascorbic acid requirement Fish meal replacement in diet Genomic regions for growth-related traits Taste preferences and feed acceptance Effect of dietary amino acids on growth, feed utilization and gene expression Utilization of different dietary carbohydrate sources
Luin et al. (2013) Mustafa et al. (2013) Chen et al. (2014)
5 10 15 20 25 30 35
0.25 0.32 0.31 0.30 0.25 0.21 0.19
0.04 0.01 0.04 0.03 0.07 0.02 0.02
Specific growth rate (% d1) 2.30 2.69 2.80 2.74 2.52 2.06 1.95
0.30 0.06 0.22 0.23 0.53 0.17 0.40
De et al. (2014) Othman et al. (2015)
Jiang et al. (2015) Jiang et al. (2016) Rahimnejad et al. (2015) Yu et al. (2016) Bunlipatanon and U-taynapun (2017) De et al. (2016) Firdaus et al. (2016) Luo et al. (2016) Koh et al. (2016) Sun et al. (2016a,b) Ebi et al. (2018) Faudzi et al. (2018) Kubota et al. (2017) Leong-Seng et al. (2017) Wu et al. (2017)
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in aquaculture systems under the tropical conditions in Malaysia and many other countries in the region. Salinity is yet another factor that influences growth and survival of the hybrid due to its effects on metabolism. Othman et al. (2015) conducted a series of experiments by treating the juvenile stages to salinities ranging from 5 to 35 ppt. Results presented in Table 2, show that the growth performance of the fish (in terms of average daily growth and specific growth rate) was significantly higher (P < 0.05) under Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
Average daily growth (g d1)
Senoo (2006) Ching and Senoo (2008) Liang et al. (2013)
10–20 ppt salinity treatments. These differences were attributed to osmoregulatory stress as indicated by the higher levels of plasma cortisol. In this salinity range, the plasma cortisol averaged less than 15 nmol/L while it spiked to 56.50 nmol/L at 5 ppt salinity and 33.54 nmol/L at 35 ppt salinity. Othman et al. (2015) further discussed the impairment of physiological processes linked to this stress by observing the food consumption in the fish. Food conversion ratio (FCR) was less than 1.5 in specimens exposed to 10–20 ppt salinity as compared to significantly higher (P < 0.05) values in other treatment groups where it reached almost 2.4. The reasons presented were reduced feed intake under sub-optimum levels of salinity and mobilization of more energy towards ionic regulation, thus leading to diversion of some energy from the growth process. If the views of Pickering and Pottinger (1995) are given credence, the minimum (5 ppt) and maximum (35 ppt) values of salinity can be considered hypotonic and hypertonic, environments, respectively for TGGG that create osmotic imbalance and metabolic disruptions. The optimum culture conditions in terms of temperature, salinity, pH and dissolved oxygen have not been thoroughly investigated for all the stages of development of TGGG. Generally, researchers have reported a range of values for the stages of hybrid grouper reared for specific experiments (Table 3). It can be deduced from the data that the optimal values of parameters for the various stages of TGGG could be: Temperature (26–30°C), salinity (30 ppt) and pH (7.5–7.8). While pH values reported in the literature are in the range 5.1–7.8, but there could be issues related to its measurement since 5.1 is too low for the hybrid or its parent species. Views expressed by Sutthinon et al. (2015) that the optimal values depend on the developmental stage of the fish can explain the variations in the observed values. To confirm the growth advantage resulting from hybridization, Bunlipatanon and U-taynapun (2017) measured the growth performance of the hybrid and compared it with that of the parent species of giant grouper and tiger 3
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Table 3 Environmental parameters used for trials on TGGG Total length (cm)
References Growth performance
Ching and Senoo (2008) Luin et al. (2013) Bunlipatanon and U-taynapun (2017) De et al. (2016) Leong-Seng et al. (2017) Ismail et al. (2018)
grouper (Fig. 1). It appears from the data that the specific growth rate as well as average daily gain in body weight in TGGG are significantly higher than these parameters in the parent species. Luin et al. (2013) sampled TGGG of the size range 60– 90 cm and established the length-weight relationship by the following equation: Log W = 4.3317 + 2.8453 log L, where, W = Body weight (g) and L = length (cm). This equation suggested that the growth was not far from the isometric value, implying that the progression in body weight vis-a-vis body length is what is expected of a healthy fish maintained in captivity. The better growth performance of TGGG compared to parent species is not an isolated case. The hybrid of E. fuscoguttatus 9 E. polyphekadion has also been observed by James et al. (1999) to grow better than the male and female parents (Table 4). It is because of this difference in growth that hybrids attain marketable size earlier than the slow-growing parent species and inherit the consumerappealing features of E. polyphekadion that make their culture more profitable. 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 Giant grouper
Figure 1 Specific Growth Rate, SGR (% day1), Weight Gain, WG (g day1). Adapted from Bunlipatanon and U-taynapun (2017).
Table 4 Growth performance of E. fuscoguttatus and E. polyphekadion and their hybrid. Adapted from James et al. (1999)
Daily growth, g Grow-out period for marketable weight (months) Average marketable weight (g)
E. E. fuscoguttatus polyphekadion 2.34 7.0
It should be emphasized that an accurate comparison of growth of hybrids with different parent species combinations would be possible if they are cultured under similar conditions since growth of the fish heavily depends on diet and rearing systems. However, because giant groupers attain a larger size, the TGGG hybrids inheriting this tendency together with their resilience will probably yield better biomass production. The physiological, molecular and genetic basis of the growth superiority of the hybrid resulting from heterosis has been investigated in detail. Worth mentioning are the findings on the expression of genes linked to growth. Sun et al. (2016b), based on their work on brain and liver, reported differential gene expression in the parent fish and the hybrid pertaining to growth hormone/ insulin-like growth factor, GH/IGF axis and the downstream signalling pathways (including protein and glycogen synthesis) that contribute to higher growth tendency of the hybrid. This identification of glycogen synthesis led to focus of studies on the skeletal muscle (the effector tissue) which is an important anatomical region for glycogen dynamics and for accomplishing fish growth and bioenergetics to support life activities. Because growth of skeletal muscle depends on many other metabolic processes such as the release of calcium, the mechanism of calcium-signalling pathway received attention as well. Sequencing of transcriptomes using RNA-seq technique and validation of the data by quantitative real-time PCR (qRT-PCR) revealed enhancement of 8 out of the 10 enzymes catalyzing glycolysis in the hybrid compared to parent species (Sun et al. 2016a). A similar pattern was observed by these authors in the form of higher level of activation of the genes involved in calcium-signalling pathway and up-regulated troponins (TnC, TnT and TnI) in the hybrid. Bunlipatanon and U-taynapun (2017) also observed expression of growth rate genes (insulinlike growth factor, IGF-I and II) in the skeletal muscle and liver. The detected gene expression was comparable to the growth pattern, with the hybrid ranking first, followed by the giant grouper and the tiger grouper. Their Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
Aquaculture performance of grouper hybrid
studies were based on samples from liver and skeletal muscle for reasons of their metabolic roles in supporting the growth process in fish. Nutrition Groupers are highly carnivorous, and require significant proportions of dietary protein and fat for normal growth. Most of the published data on nutritional trials are based on juveniles. Many investigations have suggested their protein and lipid requirements to be in the range of 40–50% and 8–16%, respectively (Teng & Chua 1978; Tuan & Williams 2007; Shapawi et al. 2014; Yu et al. 2016). TGGG is no exception to this dietary requirement. Jiang et al. (2015) elaborated the effects of dietary protein and lipid levels on growth. Jiang et al. (2016) established the optimal protein and protein-to-energy ratio required for normal growth of the hybrid. In a recent study, Rahimnejad et al. (2015) observed that the diet containing 50% protein and 14% lipid supported maximum growth of TGGG. The nutritional data on groupers and other fish species suggest that the fish consumes food to meet its energy needs (Kaushik & Medale 1994). In captive stocks, when protein level in the feed is reduced below the optimum level, the fish consumes more feed to get the required protein calories for growth and metabolism. Optimizing feed composition supports growth and helps in reducing the amount of feed which in turn has economic implications. Hybrid grouper’s nutritional requirements are different from some other species examined except perhaps in protein level which is 50% generally for most of the species of groupers in tropical regions. This includes the observations reported by Shapawi et al. (2014) for brown-marbled grouper and the parent species of the TGGG. The differences pertain to lipid and its proportion with protein. For Malabar grouper, increase in lipid to 12% improved the feed conversion ratio as well as growth rate (Tuan & Williams 2007). Increase in lipid content to 16% decreased the food conversion ratio (FCR) in brown-marbled grouper (Shapawi et al. 2014) and polkadot grouper (Williams et al. 2004). Opinion of researchers is divided on the protein-sparing role of the dietary lipid in different species of groupers. This role was established in the case of humphead grouper (Williams et al. 2004) and in brown-marbled grouper (Shapawi et al. 2014) but Tuan and Williams (2007) and Rahimnejad et al. (2015) could not find any evidence of it in the Malabar grouper and TGGG. Most likely, this could be due to differences in the efficiency of compensatory mechanisms that enable higher protein efficiency ratio (PER) in species offered low-protein diets. Earlier, Berger and Halver (1987) raised this possibility for fish. Results of the feeding trials of TGGG point to the limited ability of this Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
fish to oxidize lipid as a source of metabolic energy. Probably, in times of sustained energy needs the TGGG relies more on protein. In Asia, low-value fish caught from the wild are minced and directly fed to groupers or are processed into aquafeed containing fish meal and fish oil. Use of fish-based feeds remains a major controversial issue in aquaculture. It poses a serious problem for the oceans since the prey fish populations are declining and marine ecosystem is in a disturbed state. Costa-Pierce and Page (2010) have examined this problem and emphasized the need for accelerating research for incorporating sustainable feed ingredients in fish diets that can eliminate the need for fish meal and oil in aquaculture. Fish grow well on natural diets that provide nutrients in amounts enough to meet their metabolic requirements. The essential nutrients are amino acids, fatty acids, vitamins, minerals and energy-yielding macronutrients, namely protein, lipid and carbohydrate (Hixson 2014). Diets for fish must contain all the essential nutrients and energy to support growth and general wellbeing of the fish in aquaculture systems. Dietary protein is the source of amino acids which the fish utilizes to synthesize new proteins and to maintain the existing protein in cells and tissues while the excess quantity is mobilized for energy. Lipids supply the essential fatty acids and energy, and are structural components of cell membranes, and precursors of steroid hormones and prostaglandins. The fatty acids particularly needed by fish are the long-chain Polyunsaturated Fatty Acids (PUFA), especially linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. Dietary carbohydrates provide energy and participate in other physiological roles. Feeds that do not provide these nutrients will not support healthy living condition of the fish, and the production in aquaculture will decline. Bioavailability (the proportion of nutrients in the feed that is digested and absorbed by the fish) is also an important consideration. A carnivorous fish like the hybrid grouper has enzyme systems for protein-rich food of animal origin and might neither accept a predominantly vegetarian diet nor digest it effectively. However, it is possible to develop formulated feeds with a balance of nutrients derived from sustainable sources that can meet the physiological requirements of the fish. Tacon and Metian (2009) have discussed the urgent need for substituting the fishmeal and fish oil with alternative sources of nutrients. Some plant-based sources already being used as a partial substitute include sunflower and linseed (Naylor et al. 2009). However, they do not contain the omega-3 fatty acids. Single cell oils extracted from marine microorganisms have given good results. For instance, Miller et al. (2007) reported that thraustochytrids, a group of marine microorganisms, can be used to replace fish oil in the diet of juvenile Atlantic salmon without any apparent adverse 5
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effects on fish health. This topic is receiving increasing attention due to its importance in aquaculture. Rahimnejad et al. (2015) used a mixture of soybean oil and linseed oil in lipid-rich diet formulated for TGGG juveniles and noticed selective retention of linoleic acid in the muscle and liver. This reflects a good capacity of the hybrid grouper that can help in feed development. Given the importance of dietary protein for TGGG and the concern to explore sustainable sources of nutritional components, the feasibility of using soy protein concentrate (SPC) as an alternative to fish meal in the feed was examined (Faudzi et al. 2018). Juvenile specimens of TGGG of average body weight 6.1 0.7 g were fed six isoproteic (50% protein) and isolipidic (12% lipid) diets containing different levels of SPC as fish meal replacement, amounting to 20%, 30%, 40%, 50% and 60%, for a period of 6 weeks. Growth of the fish was highest with SPC20 and lowest with SPC60. However, there was no significant difference (P < 0.05) in growth of SPC20 treatment with SPC0, SPC30, SPC40 and SPC50 compared to growth with SPC60 treatment. The Specific Growth Rate (SGR, %day1) was in the range of 3.8–4.8, with the lowest value in SPC60. The data provided a basis to conclude that SPC is a suitable feed ingredient that can serve as a 50% replacement of fish meal. When in excess of this level, SPC reduces digestibility and feed utilization which adversely affect the growth performance of TGGG. While protein content of SPC is important in determining its nutritional efficiency in TGGG, the other factors, including the balance of essential amino acids, fatty acid profile, and anti-nutritional factors also deserve consideration. Certain qualities of SPC particularly its low contents of antigenic substances, oligosaccharides and less trypsin enzyme inhibition activity help in better protein utilization and growth-promoting effect in the fish (Peisker 2001; Wang et al. 2004). In an exhaustive study on TGGG, Ismail et al. (2018) studied the utilization of different dietary sources of carbohydrate in the hybrid grouper. These authors tested four experimental isoproteic (50% protein) and isolipidic (10% lipid) diets containing carbohydrates from different sources, namely, tapioca, corn, sago and dextrin at the same inclusion level (20%). Results of this trial (Table 5) conducted over a period of 10 weeks showed that the body weight gains were higher with tapioca, corn and sago than dextrin; net protein utilization was maximum with corn diet, followed by dextrin, sago and tapioca and food conversion ratio and protein efficiency ratio were not significantly influenced by these sources of carbohydrates. Hepatosomatic index was significantly higher with the dextrin diet. Muscle protein and lipid were higher with sago diet. These could be related to a number of factors such as texture of the pellet, digestibility and physiological tendency of the fish to process carbohydrates from different 6
Table 5 Feed utilization, growth, organ condition and proximate composition in TGGG in a 10-week trial. Adapted from Ismail et al. (2018) Parameters measures
Final body weight (g) Body weight gain (%) Specific growth rate (% day1) Food conversion ratio Protein efficiency ratio Net protein utilization Hepatosomatic index Protein (%) in muscle Lipid (%)
Experimental diets Corn
121.69 782.09 3.25 1.17 1.64 32.92 1.40 18.50 0.55
124.28 798.01 3.28 1.19 1.58 30.11 1.39 18.75 0.79
120.84 780.90 3.24 1.15 1.63 29.38 1.65 17.82 0.49
111.27 707.66 3.12 1.17 1.63 30.28 2.77 18.22 0.46
sources into other metabolites. Considering that TGGG is a carnivorous fish, its ability to use such a high proportion of dietary carbohydrate is a remarkable finding. Probably, not all sorts of carbohydrates can match that level of efficient utilization. Even among the three sources of starch, sago gave the best results in promoting growth. It emerges that the hybrid is capable of digesting a significant proportion of starch if the diet is formulated in a way that it contains appropriate amounts of other nutrients, especially protein. These findings provide an opportunity of examining more carbohydrate choices for economizing and improving the ecological aquaculture of groupers. Studies on carbohydrate content of the TGGG diet are necessary for examining the physiological efficiency to make use of a smaller proportion of this nutrient in its natural diet to formulate feeds that are more economical and have a less ecological footprint. The fact that TGGG is a cross between two highly predatory species, its diet should be predominantly protein. Dietary protein has, therefore, received more interest. Among the notable findings in this area is the work of Rahimnejad et al. (2015). The authors conducted feeding trials, each extending over a period of 8 weeks, to determine the effects of dietary protein levels on growth of juvenile TGGG averaging 2.55 0.10 g wet body weight. Results pointed to the optimum level of dietary protein for TGGG to be 50%. It is evident from the data that growth increased with the level of dietary protein from 40 to 50%, and further increase of the nutrient to 55% reduced the growth. Increase in SGR and PER was significant. The authors modulated the lipid content of the diet from 7 to 14% and noticed that the diet containing 50% protein and 14% lipid gave the best results, whereas the diet comprising 40% protein and 14% lipid produced poorest growth in the fish. It is important to point out that the alternative protein sources, especially from plants, are associated with the palatability problem in carnivorous fish (Leong-Seng et al. Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
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2017), leading to rejection of the diet even if it is balanced in nutrients. Attempts have, therefore, been made to solve this problem in the case of TGGG. Leong-Seng et al. (2016) carried out experimental trials on taste enhancers and feeding stimulants, and identified betaine as an effective ingredient. It is an amino acid commonly found in various food sources like beets, spinach and many others from where it can be easily extracted. The investigations on taste preference of the hybrid grouper were extended by these authors to include video recording of the behavioural response of the fish to feeds containing nucleoside and nucleotides. These observations revealed that Inosine-50 -monophosphate as well as Guanosine-50 -monophosphate are functional feeding stimulants at 1% concentration. There are several on-going studies on the nutrition of TGGG and most of these are focused on producing eco-friendly and nutritionally balanced feeds that are also readily acceptable to the fish. As far as micronutrients are concerned, there is a paucity of data on their effects on TGGG despite the fact that knowledge on requirements of these essential elements can greatly help in formulating diets that can stimulate growth, support normal development and bolster the immune system of the fish. However, a survey of recent literature shows interest in this topic. One of the micronutrients is ascorbic acid which is involved in promoting growth, normal skeletal development and general health (Lin & Shiau 2005; Ai et al. 2006; Chen et al. 2015). Most teleostean species are unable to synthesize it due to lack of L-gulonolactone oxidase
enzyme (Wilson 1973), and moreover, there are significant variations among the species in the amounts needed (Zhou et al. 2012; Kim & Kang 2015). Based on eight experimental diets with different supplementation levels of ascorbic acid, Ebi et al. (2018) established that the hybrid grouper requires 156 mg/kg of dietary ascorbic acid in the feed for optimum growth and development. This information is of vital importance in developing functional feeds comprising specific micro ingredients targeting different functions in TGGG to contribute to growth, immunity, resilience to environmental variations and other physiological benefits beyond those provided by general type of feeds focusing only on biomass gain in farmed fish. Triastuti et al. (2018) working on juvenile fish 1 month after hatching noticed vertebral abnormalities and suggested several causative factors, including the deficiencies of vitamins and minerals. Ebi et al. (2018) carried out a detailed study on the dietary ascorbic acid requirements while focusing their observations on growth and skeletal features of the fish over a period of 10 weeks. The experiment involved eight graded levels of ascorbic acid (4.8– 303 mg/kg). Results indicated that while the ascorbic acid content of the diet did not affect the survival but it influenced growth and skeletal development. The radiographic imaging has shown fusion, kyphosis, lordosis and scoliosis in the juvenile grouper hybrid resulting from ascorbic acid deficiency (Fig. 2). The highest specific growth rate (3.24%d1) and wet body weight (880.18%) occurred in specimens where dietary
60 50 40 30 20 10 0 C5
Figure 2 Occurrence (%) of vertebral deformities in juvenile hybrid grouper fed diets containing different levels of ascorbic acid. Adapted from Ebi et al. (2018). C5, C11, C24, C47 and C76 are diets supplemented with ascorbic acid amounts of 4.8, 11.2, 24.1, 47.2 and 75.6 mg/kg, respectively.
Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
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supplementation of ascorbic acid was 156 mg/kg. Obviously, this is the effective dose of ascorbic acid for the hybrid grouper juveniles for optimum growth rate and normal skeletal development. Attention is also being given to examining the effects of minerals on TGGG. Studies have mainly focused on Manganese (Mn), Cobalt (Co) and Zinc (Zn). Liu et al. (2016) examined the effect of Mn and Co on juvenile TGGG. Their study was based on six dietary treatments (T) of Mn supplementation amounting to 7.48 (T 1), 10.34 (T2), 13.76 (T3), 19.72 (T4), 31.00 (T5) and 53.91(T5) mg/kg. The key findings from the presented data are that: the survival was not significantly affected by these dietary treatments; specific growth rate increased from 1.62 to 1.79% day1 with increased intake of Mn from T1 to T3 and then significantly (P < 0.05) declined with further increase in the Mn dose, giving optimum body requirement of 12.70 mg/kg diet; higher FCR, and vertebral zinc and iron contents in fish fed the basal diet were linked to the fish’s physiological capacity in the form of compensatory response to Mn deficiency. Earlier, Liu et al. (2016) established the significance of dietary supplementation of cobalt methionine through growth and physiological investigations. Preliminary observations on the effect of dietary Zinc (Zn) on TGGG have been documented by Kiat (2018). He suggested 33.4 mg Zn/kg feed as the appropriate dose for the fish. While amount almost half this quantity reduced growth but excess quantities did not significantly improve the gains in body weight. Disease resistance Infectious diseases pose a serious threat to the sustainability of aquaculture, and cause economic losses to the farmers and shortage in supply of fish to the market. Treatment of the fish stocks with antibiotics is not allowed due to concerns about the health of consumers resulting from eating fish containing residues of antibiotics or other harmful chemicals, and the impact of aquaculture effluent on the environment. Pathogen exclusion from culture systems and disease resistance of the captive stocks are considered more practical measures in fish farming. Preventing entry of pathogens into the system by way of water or food can be expensive and difficult for open type aquaculture. Vaccination against bacterial pathogens has a limited scope and is used for certain high-value species of fish. There is no vaccination treatment for viral infection of fish. Obviously, inbuilt disease resistance can contribute greatly to the fullcycle aquaculture of grouper. The hybrid grouper’s higher resilience and lower mortality are the qualities that are of great aquaculture advantage. Vibriosis is a common disease of significant concern to the grouper farming industry. It is caused by bacteria belonging to the genus Vibrio. Experiments have been 8
conducted to test the resistance of TGGG against different species of Vibrio and comparing it with that of the parent species. Bunlipatanon and U-taynapun (2017) challenged TGGG with Vibrio vulnificus and examined the immunological parameters, clearance time of this pathogen and the fish survival. The authors noticed that the leucocyte number, lysozyme activity and the ability to eliminate V. vulnificus were much higher in the hybrid and giant grouper compared to tiger grouper. Furthermore, the mortality rate of tiger grouper was observed to be higher whereas its survival after 15 days post-challenge was lowest compared to the hybrid and giant grouper. Ebi et al. (2018) also investigated the susceptibility of TGGG to Vibrio harveyi VHJR7 at three different concentrations of the bacteria (8.4 9 104 (T1), 1.3 9 105 (T2), and 1.7 9 105 (T3) c.f.u. g1 body weight. Observations made over 10 days of post-challenge revealed that the cumulative mortality of hybrid grouper ranged from 0 to 57.1%. The hybrid grouper was found to be susceptible to V. harveyi VHJR7 at high doses (1.6 9 105 c.f.u g1 body weight) and the LD50 value was higher compared to the parent species. The authors attributed it to the hybrid vigour. It is important to note that stocking of the grouper at high density and culturing above 15°C enhance their susceptibility to vibrio (Bunlipatanon & U-taynapun 2017). In South-East Asia and other tropical regions, groupers are cultured under these conditions. Stocking at high density is for economic reasons while farming at temperature higher than 15°C is due to natural weather of the area. In fact the temperature range of 26– 30°C is optimum for its growth (De et al. 2014, 2016). Under these situations the importance of boosting the innate disease resistance in the grouper achieved through hybrid vigour can hardly be over emphasized. Working on growth and immune system of TGGG, Ebi (2018) established the role of vitamins C and E. After a series of trials, the author reported effective doses of vitamins C and E as 18 mg/1 kg and 815 mg/kg of feed, respectively in supporting optimum growth and good skeletal health, and also in enhancing the immune responses and disease resistance of the fish. Climate change resilience The conceptual work done on vulnerability of aquaculture to climate change (De & Soto 2009; Handisyde et al. 2017) has contributed to increasing the understanding of risks facing the fish supply. A growing number of scientific reports (Mustafa 2015; Mustafa & Shapawi 2015; Mustafa et al. 2015, 2018) have emphasized the need to reduce vulnerability and increase the adaptive capacity. In this context, replacement of marine fish-based feeds and novel aquafeed ingredients, integrated multi-trophic aquaculture systems and identifying climate resilient species are among Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
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the priority topics for climate-smart aquaculture solutions. Perhaps, the most challenging of all these areas is identifying resilience traits and species that can cope with the complexity of issues linked to changing climate. Research on this aspect is at an early stage and there is only one report (Mustafa et al. 2013) presenting results of the trial on TGGG and its parent stocks in a simulated ocean acidification. The experiment involved treatment of seawater with carbon dioxide to change the carbonate chemistry and reduce the pH in the way it is happening in climate changedriven ocean acidification. The data revealed better tolerance of the hybrid vis-a-vis parent species. Conclusions Aquaculture industry has overwhelmingly welcomed the introduction of the hybrid grouper for commercial development. Faster growth of the hybrid reduces farming period and hence the externalities required for maintaining the fish, thereby reducing the resource inputs. Disease resistance obviates the need to use harmful chemicals, and this addresses the issues linked to public health and effluents. Nutritional flexibility of the hybrid makes it possible to curtail dependence on fish-based diets and this helps in sparing the marine ecosystem. Increased supply to the market also lessens the pressure on wild stocks of groupers and the marine critical habitats from where they are caught by unsustainable methods. Further success of grouper aquaculture would depend on the ability of aquaculture industry to use new approaches that minimize the use of natural resources and maximize production efficiency for the longterm benefit of the society and the environment. It is, therefore, important to give credence to overwhelming scientific evidence and take a rational view based on all these perspectives in matters as important as seafood security. Important areas for future research on hybrids include nutritional requirements, especially micronutrients, sexual maturation and fertility, cues governing sex differentiation, possibility of backcrossing involving F1 hybrids with giant grouper, improving the survival of early larval stages and culture of hybrids of different species under similar conditions for comparative analysis of their growth performance. Acknowledgement This research was supported by the Niche Research Grant Scheme (code NRGS0004) of the Ministry of Education, Malaysia. References Ai Q, Mai K, Tan B, Xu W, Zhang W, Ma H et al. (2006) Effects of dietary vitamin C on survival, growth and immunity of Reviews in Aquaculture, 1–12 © 2018 Wiley Publishing Asia Pty Ltd
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