Sample proposal on blue crabs

Diet development for blue swimmer crab, Portunus pelagicus juveniles, with emphasis on lipids nutirition PhD Candidate: Supervisor: 1.0 Background, Aims and Significance of the Project The blue swimmer crab, Portunus pelagicus also known as sand crab, is important in commercial and recreational fisheries in Australia (Lestang et al., 2003) Catches for this species have risen substantially from 200 tonnes in 1987/88 to 740 tonnes and valued $2.2 million in 1997/98 (Kangas, 2000) Increases in the catches of commercial fishers and the high participation of recreational fishers (Williams, 1982; Kangas, 2000) reflect the value of this species to consumers and increasing market demands Hatchery culture of this species is relatively successful with good survival rates (Josileen and Menon, 2005; Romano and Zeng, 2006), providing a solid basis for the fast expansion of a farming industry for this species However, although blue swimmer crabs farming is promising, so far published information on culturing this species intensively is very limited As the emerging of aquaculture industry of the species puts a strong demands on feed used to grow them (Watanabe, 2002), a comprehensive and quantitative understanding of the nutritional requirements of the species are critical to increase production and to facilitate further expansion of the industry One of the major concerns in crustacean nutrition is lipids nutrition There is ample evidence from previous studies showing that dietary lipids imbalance severely reduced growth rates, molting frequency and survivals Much previous work has been done on crustacean lipids requirement was done on Penaied prawns, and so far there is so far no published information available on lipids nutrition for blue swimmer crabs There is clear evidence that lipids requirements of crustaceans are species-specific Therefore, systematic and quantitative studies on lipid requirements for the blue swimmer crabs are necessary Lipids consist of varying classes and constituents, which includes phospholipids, fatty acids, sterols and caratenoids (L.Gonzalez-Felix et al., 2002) Each of these components needs to be supplied in optimum amount and balanced with other constituents Phospholipids, normally supplemented in the form of lecithin in formulated feed, and cholesterol, are bio-membranes components that are important in maintaining cellular functions and structure (Gurr and Harwood, 1991) It is believed that lecithin facilitates transport of cholesterol and has significant interactions when supplied together in the diets (Gong et al., 2000) However, few studies have focused on their interactions As a cell component and a metabolic precursor of steroid, brain, vitamin D and molting hormones (New, 1976; Gong et al., 2000; Hernandez et al., 2004), dietary cholesterol is also essential for the growth of crustaceans Sargent (1999) pointed out that, essentiality of highly unsaturated fatty acids (HUFA), ecosapentaenoic fatty acids, (EPA; 20:5n-3), docosahexaenoic fatty acids (DHA; 22:6n-3) and arachidonic acids (ARA; 20:4n-6), and competitive interactions between them, are fundamentals to lipids nutrition study Crustaceans need supplemental dietary HUFA because of their inability to elongate and desaturate polyunsaturated fatty acids (PUFA) to HUFA (L.Gonzalez-Felix et al., 2002) Much research has been done on n-3 HUFA requirements in crustacean; however, less information is available on the important of optimal ratio of EPA to DHA Because both DHA and EPA play major roles in survival and growth of crustaceans, it is possible that n-3 HUFA requirements are not only a function of total amount but also the proportions of EPA and DHA Furthermore, less attention of the past dietary HUFA investigation in crustaceans has been given to arachidonic acid as essential fatty acids (EFA) Eicosanoids that are produced from ARA are more biologically active than those produced from EPA (Sargent et al., 1999) and it plays important roles in crustaceans molting (Sheen and Wu, 1999) Although it has been reported, removing ARA from P monodon diets resulted in no notable response in growth rate (Merican and Shim, 1996), conservation of ARA during starvation signifies some important functions of ARA (Wen et al., 2006) Lack of research on ARA requirements in crustaceans is believed a major attributor for the uncertainty of nutritional values of ARA as a major HUFA for crustaceans and further investigation is needed Nutrition is one of the core factors in blue swimmer crab cultivation and needs to be explored in great details for further development of this species A good formulated diet not only will boost the productions of cultured species, but will also help maintaining water quality Particle break downs from diets has been suggested to cause deterioration of water quality (Meyers et al., 1972) However, water stability of the diets can be increased by using binders (New, 1976) that helps form compact and durable pellet (Silva and Anderson) Still, binders may also impair digestibility of feed and loss of nutritional content due to high leaching rate of water soluble nutrients (Meyers et al., 1972; New, 1976; Genodepa, 2004) Although some nutrients leaching may be necessary to stimulate feed intake (Baskerville-Bridges and Kling, 2000), the balance between leaching rate and amount of nutrients left for culture animals to consume is far more important Nutrient levels and water stability are not the only important factor in developing formulated diets The quantity of food consumed also has pronounced effects on growth rate, efficiency of food conversion and chemical composition (Reinitz, 1983) of culture organisms In addition, this factor is also water correlated with the size of culture organism and water temperature (Khan et al., 2004) The significant difference of food ration and temperature is mostly important in region where there are noticeable changes in temperatures (e.g winter and summer) where the amount of food can be adjusted to suit culture organism requirements As optimization of feeding rate of culture animals are important to achieve efficient production (Khan et al., 2004), the final stage of this study will focus on finding the right ration for blue swimmer crabs at different water temperatures In summary, the main aims of the proposed PhD project are to quantify the optimum amount of dietary lipids and its constituents that are needed for the early blue swimmer crab juveniles The research outcomes will develop formulated diets for blue swimmer crabs and will have good potential of being commercialized A quantitative and qualitative approach will be used to achieve the following objectives: To investigate variation on lipids composition of the early P pelagicus juveniles under normal feeding and various starvation condition; To determine total dietary lipids requirements of P pelagicus juveniles; 3 To evaluate potential interactions of lecithin and cholesterol at different dietary levels; To determine n-3 HUFA requirements of P pelagicus juveniles; To evaluate potential interactions of DHA (22:6n-3) and EPA (20:5n-3) at different dietary level; To determine n-6 HUFA, arachidonic acid requirements of P pelagicus juveniles; To optimize binder component for diets fed to P pelagicus juveniles; and To investigate effects of water temperature and ration on survival and growth of P pelagicus juveniles, in individual and communal culture 2.0 Research Plan 2.1 2.1.1 General Materials and Method Source of experimental crabs Juvenile blue swimmer crabs to be used in all experiments will be reared in laboratory Wild caught broodstocks will be maintained in 1000 L outdoors tanks until spawning Berried females will be transferred indoor and kept in 300 L tank Upon hatching, larvae will be cultured with rotifers and Artemia until settlement as fist stage crabs (C1) based on protocols established at JCU (Romano and Zeng, 2006) 2.1.2 Experimental setups As cannibalism could substantially affect the results experiments, experiments to will be conducted by housing individual crabs in 750 ml perforated plastic containers (7.5 cm diameter and 12 cm height) The containers will be labeled for treatments and will be randomly distributed in water baths for temperature control The experiments are expected to be carried out mainly from crab stage one to five (C1 to C5) Except for Experiment and 8, a minimum number of 20 individually kept crabs will be used for each treatment and the treatment will be triplicated Experimental crabs will be fed every morning until satiation and any food leftover will be siphoned out before each feeding Mortality and molting will be recorded daily 100% of water changes will be conducted every day Once per week, DO, pH and ammonia level will be checked to ensure good water quality Temperature, salinity and pH will be maintained at 28oC, 30 ppt and 8.5, respectively Throughout the experiments, photoperiod will be set at 12h ratio (light:dark) 2.2 Diet Preparation Except for experiment 1, formulated food will be prepared and the ingredient of the basal diet as listed in Table will be used except some necessary modifications in starch and cellulose levels to maintain isocaloricity of the diets used in the same experiment Before preparations of the diets, lipids will be extracted from basal ingredients using hot ethanol to minimize the contribution of other dietary lipids (Sheen 2000) The feeds will be crumbled and sieved to get desired size (< 2mm) It will be stored at -20oC to prevent rapid oxidation of lipids and remain the quality of the diets To obtain persist and reliable results in growth trial, prior to experiments commence, the diets will be tested for their stability in water and crabs acceptability Table 1: Basal mix of diets (modified from Sheen and Wu 1999; 2000) Ingredient Defatted squid meal Vitamin mixturea Mineral Mixtureb Choline chloridec DCPd Zein e Source Skretting Tasmania Rabar Pty Ltd Rabar Pty Ltd Sigma-Aldrich Pty Ltd Sigma-Aldrich Pty Ltd Sigma-Aldrich Pty Ltd Total a) % of dry weight 50% 4 0.6 62.6% ZZ603 DO 067 DPI, each 1kg contains: copper 1g, cobalt 100mg, magnesium 59.4mg, manganese 5g iodine 800mg, selenium 20mg, iron 8mg, zinc 20g, aluminium 100mg, chromium 100mg b) ZZ600 DPI, each 1kg contains: vitamin A 2miu, vitamin D3 0.8miu, vitamin E 40g, vitamin K 2.02g, inositol 50g, vitamin B3 30.40g, vitamin B5 9.18g, vitamin B9 2.56g, vitamin B2 4.48g, vitamin B12 0.004g, biotin 0.1g, vitamin B6 4g, vitamin B1 3.4g, vitamin C 44.4g, para amino benzoic acid 20g, tixosil 5g, antioxidant 30g c) 98% powder C7527 d) dibasic calcium phosphate C4131 e) Z3625 from maize 2.3 Proximate and Fatty Acid Analysis of Diets and Crabs The diets and sample of pooled whole crabs at the beginning and termination of the experiment can be analyzed for proximate compositions and fatty acids based on the AOAC (1984) methods (Sheen and Wu, 2003) Crude protein will be determined using Kjedahl method while total lipid content in the samples will be determined by the chloroform-methanol (2:1, v/v) extraction method (Folch et al 1957) Ash and moisture can be measured using muffle furnace and laboratory oven Fatty acids analysis can be done based on modified method reported by Sheen and Wu (1999) 2.4 Data Collection For all experiment carapace length and width, wet weight and survival of crabs will be recorded for each molt Samples will also be taken for histology and at the end of experiments for dry weight measurement Depending on experimental design, either one-way or two-way ANOVA will be used for data analysis with Tukey’s test to identify significant differences among treatments Experiment 1: Effects of starvation on growth, survival and tissue biochemical and fatty acid profiles of blue swimmer crabs early juveniles Rationale Effects of starvation on utilization of nutrients have been studied in crustaceans to gain more understandings on their requirements (Barclay et al., 1983; Clifford and Brick, 1983; Dall and Smith, 1986; Wen et al., 2006) A study conducted on juvenile Chinese mitten crab showed that PUFA and HUFA were conserved in both hepatopancreas and muscle of the crab at the expense of saturated and monounsaturated fatty acids (Wen et al., 2006) It has been proved that conservation of PUFA and HUFA are because of their importance as cell structure Outcomes of this experiment will help understanding the preferential of lipid constituent conserved during starvation for blue swimmer crabs and thus provide useful clues on lipids requirements of this species Methodology A total of 656 juvenile crabs will be used and will be divided to treatments, i.e fed (F), starved (S) and starvation for days and re-fed (7 S-F) Crabs will be fed with commercial pellet produced by Ridley® for the tiger shrimp, Penaeus monodon, which contains 43% protein, 6% fat and 3% fiber Samples will be taken times for all treatments during the experiment for proximate and fatty acids analysis as well as histology Table 2: Time when sample will be taken Treatment 3rd day Fed (F) Starved (S) Day Starvation and Re-fed (7 S-F) 7th day ▪ ▪ ▪ ▪ 10th day C2 C3 ▪ ▪ ▪ ▪ ▪ Expected Outcomes • Data on survival, development and growth as well proximate and fatty acid profile and histochemistry of digestive gland will be used to assess preferentially conserved and utilized of major nutrients and lipids in both fed and those under various starvation conditions Experiment 2: Effects of various dietary lipid levels on the survival, development and growth of the blue swimmer crabs juvenile Rationale Levels of lipids in the diets are closely associated with crustaceans survival, development and growth, thus there are substantial interest in determining the optimal total lipids that a particular crustacean needs (Sheen and Wu, 1999) For crustaceans, both inadequate and excessive lipid levels negatively impact their grow-out Low level of dietary lipids may result in low molting frequency (Sheen and Wu, 1999) and at high levels, retarded growth (Ackefors et al., 1992) Requirements of total lipids for crustaceans seem to be species-specific, although generally it is within 5-10% range (Sheen and Wu, 1999) Methodology Seven diets with lipid levels ranging from 0-12% at 2% increments and 2:1 ratio (fish oil:corn oil) (Sheen and Wu, 1999) will be formulated and used to feed crab juveniles For the production of crabs for the experiment and other experimental design, data collection, please refer to ‘General materials and method’ (subsection 2.1) Expected outcomes • Established optimal total lipids levels in the diets for the blue swimmer crabs juveniles Experiment 3: Determining optimum levels and ratios of dietary cholesterol and lecithin and their effects on survival, growth performance and tissue lipid composition for blue swimmer crab juveniles Rationale Lecithin play an important role in lipid mobilization and is believed that it facilitates transport of cholesterol (Gong et al., 2000) Cholesterol is a compound of sterols and important as a cell component and is a metabolic precursor of brain, vitamin D and molting hormones (New, 1976; Gong et al., 2000; Hernandez et al., 2004) It has been proven that imbalanced cholesterol and lecithin in the diets negatively impact crustacean growth (Chen and Jenn, 1991) This highlights the need to investigate balanced supply of cholesterol and lecithin in the blue swimmer crab diets Methodology Three level of cholesterol of 0%, 0.5% and 1.0% will be paired with three levels of lecithin, 2%, 4% and 6% Levels of both cholesterol and lecithin were chosen based on literature on range required for crustaceans All nine diets will be evaluated based on survival and growth performance of blue swimmer crabs juvenile Effects of levels of dietary cholesterol and lecithin on tissue fatty acids composition will also be investigated Expected outcomes • The optimal level of lecithin and cholesterol for the blue swimmer crab juveniles will be determined and used as the basis for formulating diets in following experiment • Understanding the potential compensatory effects of lecithin and cholesterol Experiment 4: Determining the optimal levels of n-3 HUFA for blue swimmer crab juveniles Rationale n-3 highly unsaturated fatty acids (n-3 HUFA) that contains eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), plays important physiological roles as components of membrane phospholipids and as precursors of biologically active eicosanoids (Bell et al., 1986; Mourente and Tocher, 1992) Due to their various function and inability of crustaceans to synthesize their own n-3 HUFA, inclusion of n-3 HUFA in most favorable amount is essential to boost good survival and growth Although, the importance of n-3 HUFA to crustaceans species are well documented, the requirements of n-3 HUFA for blue swimmer crab is unknown Methodology For this experiment, six diets containing levels of n-3 HUFA from to 2.5% with a 0.5% increment will be formulated Inclusion of cholesterol and lecithin in all diets will be based on best interaction levels of these compounds in previous experiments Fish oil will be used as source of n-3 HUFA and will be adjusted to desired n-3 HUFA levels that will be tested in this experiment Expected Outcomes • The optimal level of n-3 HUFA in the diets of blue swimmer crabs juvenile will be determined Experiment 5: Identifying optimal ratio of EPA (20:5n-3) and DHA (22:6n-2) for blue swimmer crab juveniles Rationale Among the numerous study on n-3 HUFA requirements for crustaceans, relative little attention have been given to address requirements of EPA and DHA individually (Read, 1981; Merican and Shim, 1996) Of those relative few research, most of it often focused on DHA requirements (Merican and Shim, 1997; Mourente and Rodriguez, 1997) Suprayudi (2004) suggested that DHA is more efficacious than EPA in promoting growth However, the fact that EPA is important in maintaining survival and involved in producing eicosanoids (3-series prostanoids and 5-series leukotrienes) (Sargent et al., 1999) indicating that both of the fatty acids are important and balanced proportions of these compound might be crucial Study on P monodon illustrated adverse effects on weight gain when fatty acids ratio was imbalanced (Glencross et al., 2002b) The study showed that best growth was obtained when same amount of EPA and DHA were supplied Methodology Total level of n-3 HUFA in this experiment will be based on the best result in n-3 HUFA experiment (Exp 4) However, in formulating the diets, EPA and DHA will be adjusted to match the tested ratio Source of EPA and DHA will be from purified blended n-3 HUFA fish oil All seven diets will be fed to blue swimmer crab juveniles following protocols described in General Materials and Methods (subsection 2.1) Table 3: Percentage of EPA and DHA and their ratio in tested diets EPA (%) 100 50 35 65 25 75 DHA (%) 100 50 65 35 75 25 Ratio (EPA:DHA) 0:1 1:0 1:1 1:2 2:1 1:3 3:1 Expected Outcomes • Determine the optimum dietary EPA and DHA ratio for blue swimmer crab juveniles Experiment 6: Determining the optimal levels of arachidonic acid, ARA (20:4n-6) for blue swimmer crab juveniles Rationale Molting process in crustaceans are regulated by eicosanoids substances derived from ARA (L.Gonzalez-Felix et al., 2002) When Penaeus esculentus when injected with prostaglandin E2, a type of eicosanoids, shorter molt cycles and better growth were displayed compared to control group (Koskela et al., 1992) As a HUFA constituent, ARA also could not be internally synthesized by crustaceans and need to be provided through the diets Lack of n-6 fatty acids in the diet have been proven to have negative effects on growth (Reigh and Stickney, 1989; Glencross and Smith, 1999) Despite the important of ARA to crustaceans, only limited information on quantitative requirements of ARA are available 10 Methodology Six diets will be formulated containing ARA from to 2% with 0.5% increments All diets will be formatted to contain optimum level of n-3 HUFA and best ratio of EPA and DHA based on experiments and Expected outcomes • Optimum level of ARA requirement for blue swimmer crab juveniles • Understanding the effects of n-6 HUFA and n-3 HUFA on blue swimmer crab juveniles Experiment 7: Optimizing binder compound in formulated diets fed to blue swimmer crab juveniles Rationale Various studies have assessed the use of various binders in formulated diets for aquaculture animals (Knauer et al., 1993; Pearce et al., 2002; Genodepa, 2004; Ruscoe et al., 2005) Inclusion of binder is necessity to ensure water stable pellets (Cuzon et al., 1994) Stability of the feeds in water is especially important for crustaceans which generally find food by chemoreception rather than sight, and may not consume it for several hours (D'Abramo, 1997) However, some binding agents were claimed to impair the digestibility of animal feeds (New, 1976) and may also results in diets that are not adequately stable and cause excessive nutrient loss and water pollution (Genodepa, 2004) Methodology Diets with optimum levels of lipids, based on previous experiment, will be formulated with different types of binders; zein, gelatin, alginate and agar at the same level (3%) (Genodepa, 2004) The diets will be evaluated by measuring physical parameters of the diets in seawater column (water stability and leaching rate) and nutritional performance of the diets (survival and growth) Water stability of the diets will be based on modification of method used by Ruscoe et al (1995) Dry matter remaining (DMR) of the diets after interval periods of immersion will be determined DMR will be determined using following equation: DMR (%) = Wo x (1-M) –Wt x100 Wo X (1-M) 11 Wo = initial diet weighed, Wt = diet weight after immersion and drying, M = moisture content of diet as a proportion Leaching rate of the diets will be evaluated using methods proposed by Baskerville-Bridges et al., (2000) Diets with different binders will be formulated separately by adding free amino acid, glycine Glycine via leaching after various intervals immersion will be calculated using ninhydrin reagent and spectrophotometer Diets without any addition of binder will be used as a control and will be tested in the same way Table 4: Time when sample will be taken Diet Immersion Period Test Water stability Leaching rate 15 30 ▪ ▪ ▪ 60 2h 3h 6h 12h 18h 24h ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ Diets with different binders will be fed to crabs based on feeding methods used in previous experiments Diets performance will be determined on the basis of survival and growth Expected outcomes • Water stability of the diets at different time immersion • Leaching rate of diets with different binders • Crabs acceptance toward the diets with different binders • Best binder will be used to formulate diets for next experiment Experiment 8: Effects of water temperature and food ration on growth performance of blue swimmer crab juveniles, in individual and communal culture Rationale Optimization of feeding rate of culture animals is important to achieve efficient production (Khan et al., 2004) and temperature is among important factors that controlled growth of crustaceans (Wyban et al., 1995) Temperature directly effects the rate of physiological process (Spanopoulous- 12 Hernandeza et al., 2005) and thus elevated metabolic rate of crustaceans at higher temperatures (Allan et al., 2006) This is parallel with increased in feeding rates at increased water temperature levels in P vannamei juveniles (Wyban et al., 1995) Information on food ration and temperature manipulation will be useful in culturing blue swimmer crabs at different time of the year Methodology Diets in this experiment will be formulated based on previous results It will be then fed to crabs at different feeding rates, (5%, 10%, 15% and 20% of wet weight) and two different temperatures; 22oC and 30oC as in winter and summer condition Crabs will be cultured individually and also communally in twenty four 60 L perforated containers The containers will contains 20 crabs each and will be submerged in eight 300 L tanks with three containers placed randomly in each tank Crabs will be individually and communally culture to determine growth and survival rate between two culture conditions Metabolic rate of the crabs will be determined using a modified method used by Allan (2006) to verify if there any changes in metabolic rate at tested temperatures Crab will be culture individually in 100 ml conical flask sealed with lid and petroleum jelly for h at tested temperature Control flask will be prepared in the same way without any crab The oxygen concentration between control and tested flask and crab’s wet weight will be measured using DO oxygen meter and electronic balance after h Mass specific oxygen consumption rates of the crabs will be expressed as microliter of oxygen consumed per milligram of wet weight per hour (µl O2 mg wet weight-1 h-1) Expected Outcome • Best food ration for blue swimmer crabs at different water temperatures • Growth and survival rate differences between individually culture and communally culture crabs • Metabolic rate changes at different temperature 13 3.0 Timeline Tasks 06 A S O 07 N D J F M A M 08 J J A S O N Writing Literature Review Proposal Thesis Research Exp Data Analysis Exp Data Analysis Exp Data Analysis Exp Exp Exp Data Analysis Exp Data Analysis Exp Data Analysis 14 D J F M A M 09 J J A S O N D J F M A M J J 4.0 Budget Broodstock food……………………………………………… $ 1,200 Equipment…………………………………………………… $ 1,000 Chemical……………………………………………………… $ 200 Diet ingredients……………………………………………… $ 2,000 Analysis……………………………………………………… $ 6,400 Estimated Cost of Project…………………………………………… $10,800 5.0 Justification of Budget Broodstock food: The price of mussel, squid and prawns need for feeding crabs for one month is estimated at $ 100, for total years of study, $ 3,600 The amount will be shared with two other post graduate student working on crab species, $ 3,600/3 = $ 200 Equipment: The amount is needed for plastic containers, mesh, heater for crabs culture and vial, polysterene box for samples collecting and storage Chemical: Antibiotics, chlorine, alcohol and formalin will be used mainly for sterilizing laboratory and keeping the crabs from any parasite infections Diet ingredients: High quality diets will be bought from Sigma-Aldrich Company Pty Ltd, Rabat Pty Ltd, Skretting Tasmania and health store Analysis: One analysis cost $ 100, a mean of analysis per experiment will be needed for each experiment, $ 100*8*8 = $ 400 15 6.0 Publications Plan Based on experiments that will be done in this study, potential publications can be developed and published during years of study: Nutritional requirements and starvation resistance in juvenile blue swimmer crabs, Portunus pelagicus Effects of dietary lipid on growth performance and body lipid composition of blue swimmer crabs (Portunus pelagicus) Combine effects of dietary lecithin and cholesterol on the growth, survival and body lipid of marine crabs, Portunus pelagicus at juvenile stage Essential fatty acids in the diet of blue swimmer crabs juvenile (Portunus pelagicus): effects of n-3 HUFA Essential fatty acids in the diet of blue swimmer crabs juvenile (Portunus pelagicus) : effects of EPA/DHA ratio Essential fatty acids in the diet of blue swimmer crabs juvenile (Portunus pelagicus) : effects of n-6 HUFA, Arachidonic acid (20:4n-6) Effects of binder on formulated diets fed to blue swimmer crabs Portunus pelagicus The effects of water temperature and food ration on growth and survival of blue swimmer crabs juvenile (Portunus pelagicus) References: Ackefors, H., J D Castell, et al (1992) "Standard experimental diets for crustacean nutrition research II Growth and survival of juvenile crayfish Astacus astacus fed diets containing various amounts of protein, carbohydrate and lipid." 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Aquaculture 179: 217-229 Sheen, S.-S and S.-W Wu (1999) "The effects of dietary lipid levels on the growth response of juvenile mud crab Scylla serrata." Aquaculture 175: 143-153 Sheen, S S and S W Wu (2003) "Essential Fatty Acid Requirements of Juvenile Mud Crab, Scylla serrata (Forskal, 1775) (Decapoda, Scyllaridae)." Crustaceana 75(11): 1387-1401 Silva, D S and T Anderson Fish Nutrition in Aquaculture London, Chapman and Hall Spanopoulous-Hernandeza, M., C A Martinez-Palasciosb, et al (2005) "The combined effects of salinity and temperature on the oxygen consumption of juvenile shrimps Litopenaeus stylirostris (Stimpson, 1874)." Aquaculture 244: 341-348 Watanabe, T (2002) "Strategies for further development of aquatic feeds." Fisheries Science 68: 242-252 Wen, X., L Chen, et al (2006) "Effect of feeding and lack of food on the growth, gross biochemical and fatty acid composition of juvenile crab, Eriocheir sinensis." Aquaculture 252: 598607 19 Williams, M J (1982) "Natural food and feeding in the commercial sand crab Portunus pelagicus Linnaeus, 1766 (Crustacea : Decapoda : Portunidae) in Moreton Bay, Queensland." Journal of Experimental Marine Biology and Ecology 59(2-3): 165-176 Wyban, J., W A Walsh, et al (1995) "Temperature effects on growth, feeding rate and feed conversion on the Pacific white shrimp (Penaeus vannamei)." Aquaculture 138: 267-279 20 [...]... Information on food ration and temperature manipulation will be useful in culturing blue swimmer crabs at different time of the year Methodology Diets in this experiment will be formulated based on previous results It will be then fed to crabs at 4 different feeding rates, (5%, 10%, 15% and 20% of wet weight) and two different temperatures; 22oC and 30oC as in winter and summer condition Crabs will... publications can be developed and published during 3 years of study: 1 Nutritional requirements and starvation resistance in juvenile blue swimmer crabs, Portunus pelagicus 2 Effects of dietary lipid on growth performance and body lipid composition of blue swimmer crabs (Portunus pelagicus) 3 Combine effects of dietary lecithin and cholesterol on the growth, survival and body lipid of marine crabs, Portunus... 100 ml conical flask sealed with lid and petroleum jelly for 1 h at tested temperature Control flask will be prepared in the same way without any crab The oxygen concentration between control and tested flask and crab’s wet weight will be measured using DO oxygen meter and electronic balance after 1 h Mass specific oxygen consumption rates of the crabs will be expressed as microliter of oxygen consumed... the diet of blue swimmer crabs juvenile (Portunus pelagicus): effects of n-3 HUFA 5 Essential fatty acids in the diet of blue swimmer crabs juvenile (Portunus pelagicus) : effects of EPA/DHA ratio 6 Essential fatty acids in the diet of blue swimmer crabs juvenile (Portunus pelagicus) : effects of n-6 HUFA, Arachidonic acid (20:4n-6) 7 Effects of binder on formulated diets fed to blue swimmer crabs Portunus... individually and also communally in twenty four 60 L perforated containers The containers will contains 20 crabs each and will be submerged in eight 300 L tanks with three containers placed randomly in each tank Crabs will be individually and communally culture to determine growth and survival rate between two culture conditions Metabolic rate of the crabs will be determined using a modified method used by... L Harwood (1991) Lipid Biochemistry An introduction Fourth edition London, Chapman and Hall 17 Hernandez, P V., M A Olvera-Novoa, et al (2004) "Effect of dietary cholesterol on growth and survival of juvenile redclaw crayfish Cherax quadricarinatus under laboratory conditions." Aquaculture 236: 405-411 Josileen, J and N G Menon (2005) "Growth of the blue swimmer crab, Portunus pelagicus (Linnaeus,... (2000) Synopsis of the biology and exploitation of the blue swimmer crab, Portunus pelagicus Linnaeus, in Western Australia Fisheries Research Report 121, Fisheries Western Australia Khan, M A., I Ahmed, et al (2004) "Effect of ration size on growth, conversion efficiency and body composition of fingerling mrigal, Cirrhinus mrigala (Hamilton)." Aquaculture Nutrition 10: 47-53 Knauer, J., P J Britz, et al... Justification of Budget Broodstock food: The price of mussel, squid and prawns need for feeding crabs for one month is estimated at $ 100, for total 3 years of study, $ 3,600 The amount will be shared with two other post graduate student working on crab species, $ 3,600/3 = $ 1 200 2 Equipment: The amount is needed for plastic containers, mesh, heater for crabs culture and vial, polysterene box for samples... prawn Penaeus monodon " Aquaculture Nutrition 5: 53-63 Glencross, B D., D M Smith, et al (2002b) "Optimising the essential fatty acids in the diet for weight gain of the prawn, Penaeus monodon " Aquaculture 204: 85-90 Gong, H., A L Lawrence, et al (2000) "Lipid nutrition of juvenile Litopenaeus vannamei I Dietary cholesterol and de-oiled soy lecithin requirements and their interaction." Aquaculture... monodon." Aquaculture 157: 277-295 Merican, Z O and K F Shim (1996) "Qualitative requirements of essential fatty acids for juvenile Penaeus monodon." Aquaculture 147(3-4): 275-291 Meyers, S P., D P Butler, et al (1972) "Alginates as binders for crustacean rations." Prog Fish Cult Vol 34: no 1 Mourente, G and A Rodriguez (1997) "Effects of salinity and dietary DHA (22:6n-3) content on lipi composition