Towards development of a formulated diet for mud crab (Scylla serrata) larvae, with emphasis on lipid nutrition

Holme, May-Helen (2008) Towards development of a formulated diet for mud crab (Scylla serrata) larvae, with emphasis on lipid nutrition. PhD thesis, James Cook University.

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Crabs of the genus Scylla, commonly referred to as mud crabs, are commercially important crabs with an Indo-Pacific distribution. Scylla serrata is the most widespread of the four recognized Scylla species, and it has traditionally been an important fishery in coastal communities throughout its range. In recent decades it has also become a targeted species for aquaculture, however, due to a lack of low cost formulated diets, mass production of mud crab seed stock is not reliable, and the majority of mud crab farms rely on juveniles caught in the wild. This has lead to a widespread seeds-stock shortage and over-exploitation of wild populations. Development of more effective hatchery techniques and more reliable production of juveniles are therefore considered critically important for sustainable growth of the mud crab farming industry in this region.

Current methods for Scylla hatchery production rely on live foods such as rotifers and Artemia. These prey species are costly to produce, have an inconsistent nutritional profile and lack certain nutritional components essential for normal growth of marine larvae. Development of a nutritionally optimised diet is therefore considered critically important, and recent research at James Cook University has shown that mud crab larvae readily accept microbound diet (MBD) particles. As the contents of such diets easily can be manipulated, MBD shows enormous potential as a tool for further specification of the nutritional requirement of mud crab larvae.

Lipids are required in crustacean diets as an important source of energy, essential fatty acids, sterols, phospholipids and fat-soluble vitamins. An appropriate supply of these nutrients is particularly critical during larval development, where a series of important morphological, physiological and biochemical changes take place. On this basis, this thesis was designed to first collect and review all information on the nutritional requirements of S. serrata larvae published to date, and highlight areas where more research is required. Experiments were then conducted to fill gaps in our current knowledge on larval dietary lipid requirements. Some of the most important aspects of lipid metabolism; dietary cholesterol requirements, interaction between cholesterol and phospholipids, the optimum balance of dietary n-3 and n-6 fatty acids, as well as lipid class and fatty acid utilization by developing larvae have been addressed in separate chapters, with the aim to provide information assisting formulation of more appropriate species-specific MBD for S. serrata larval culture.

While previous experiments have demonstrated that megalopae of the mud crab can be cultured exclusively on MBD, the potential of MBD as a food source for zoea larvae of the same species has not previously been determined. The first experiment of this thesis was therefore set up to investigate the effect of partial and total replacement of live food with MBD for zoeal larvae, with the aim to determine the best suited larvae stage for further nutritional studies. Zoea III larvae were cultured communally at a density of 25 larvae L-1 and were fed either 100% live Artemia nauplii, 100% MBD or a 50%:50% combination of MBD and Artemia nauplii. Highest survival (66%) and development rate to the zoea IV stage were recorded for larvae fed the 50%:50% combination of MBD and Artemia nauplii. Some successful molts were also found among larvae fed exclusively on MBD, while total mortality was observed in the unfed control. The results indicate that the experimental MBD may contain certain beneficial nutrients lacking in Artemia nauplii, and that co-feeding the MBD with Artemia nauplii enhanced larval survival and development. However, the findings also suggested that total replacement of live food with the experimental MBD will result in poor survival of zoea III larvae, and based on these data, the megalopa stage was chosen as the most appropriate larval stage for further studies.

The experimental MBD previously used for rearing S. serrata megalopa contained a high proportion of dried rotifers (38% of total diet dry weight). Although this work demonstrated that mud crab megalopa readily ingest and assimilate formulated diet particles, it did not reduce the reliance on live food cultures. Fundamental to the success of this study was therefore to replace the rotifer components with a commercially available protein source, and four marine animal meals (rotifer meal, Artemia meal, fish meal and squid meal) were evaluated based on larval survival, dry weight and carapace weight of newly molted first stage crabs (C1) and development time from megalopa to C1. Fifteen megalopae were reared individually in 250 mL aquaria, and survival of MBD-fed megalopae to C1 ranged from 46.7% to 60.0% with those fed MBD containing fish meal or squid meal showing higher survival than those fed MBD containing dried Artemia or rotifers. Larvae fed live Artemia nauplii showed the highest overall survival (80%), while none of the unfed megalopae survived to C1. There were no significant differences (P >0.05) in the average time required for megalopae to reach the first crab stage when fed any of the four MBD, however, shortest development time was recorded for larvae fed live Artemia nauplii. The results indicate that squid meal is a suitable protein source for formulation of MBD for S. serrata megalopae, and this meal was used in MBD formulated for subsequent experiments.

Cholesterol is an important sterol, serving as a precursor for many physiological compounds such as sex and molting hormones, adrenal corticoids, bile acids and vitamin D. As crustaceans are incapable of de novo production of cholesterol, dietary cholesterol is essential for optimum growth and high survival in crustaceans. Chapter 4 of this thesis reports on an experiment assessing the effects of dietary cholesterol levels on growth, development time and survival of mud crab megalopae. Five semi-purified, iso-nitrogenous and iso-energetic MBD containing cholesterol levels from 0.14% to 1.00% diet dry weight were tested, using fifteen individually reared megalopae for each dietary treatment. The results showed that megalopae from all treatments were able to metamorphose to the first crab stage, suggesting that the endogenous level of cholesterol in the basal diet (0.14%) was sufficient to meet dietary requirements.

Highest overall survival (74.3%) was recorded for megalopae fed a diet containing 0.80% cholesterol, while the widest mean carapace width (3.53 ± 0.08 mm) and highest mean dry weight (21.11 ± 2.22 mg) were recorded for juveniles molting from megalopae fed live Artemia. No megalopae in the unfed control treatment metamorphosed into crabs. The average development time from megalopa to the C1 stage varied among the treatments, with megalopae fed live Artemia or MBD containing 0.20%, 0.40% or 0.80% total cholesterol showing relative shorter development time to C1; between 8.0 (± 1.46) days and 9.9 (± 1.37) days, whereas the longest development time was recorded for the megalopae fed diets containing 0.14% or 1.00% total cholesterol; 11 (± 1.45) days and 11 (± 1.24) days, respectively. Based on these findings, 0.80% total dietary cholesterol was suggested as optimum in semi-purified diets for S. serrata megalopa.

For some marine crustaceans, an interaction occurs between dietary cholesterol and dietary phospholipid. This has important implications for diet formulation, and to determine if this interaction is present in S. serrata larvae, the experiment reported in Chapter 5 was conducted to evaluate the effects of varying dietary levels of lecithin and cholesterol on growth, development and survival of megalopa. Six semi-purified MBD were formulated to be iso-energetic and iso-nitrogenous and to containing three levels of supplemental lecithin (0.0, 2.0 and 4.0% diet dry weight) and two levels of supplemental cholesterol (0.0 and 0.7% diet dry weight). The experiment was designed in the same manner as in Chapter 4, and the results showed a significant interaction between supplemental dietary lecithin and cholesterol for final mean dry weight of newly settled crabs. Highest survival (60%) was recorded for megalopae fed diets containing the highest levels of dietary lecithin regardless of whether diets were supplemented with cholesterol, and this rate of survival was identical to that of megalopae fed live Artemia nauplii. The experiment indicated that supplemental dietary cholesterol may not be essential for mud crab megalopae when fed a diet containing fish oil and sufficient levels of supplemental dietary phospholipids. Most crustacean diets today are formulated to contain a 2:1 fish oil/corn oil ratio based on known requirements of penaeid prawns. As the results from the previous chapters indicated that the nutritional requirements of S. serrata larvae are different from those of other marine crustaceans, the experiment in Chapter 6 was designed to examine the optimum fish oil/corn oil ratio in semi-purified diets formulated for mud crab megalopae. Six iso-energetic and iso-nitrogenous MBD containing 6% total lipid were formulated to contain fish oil and corn oil either singly or in various ratios (0:1, 1:2, 2:1, 3:1, 1:0, 1:1), and each dietary treatment consisted of 20 individually reared megalopae. Survival, growth, development time to C1 and signs of molting death syndrome (MDS) were recorded daily, and carapace width and dry weight of newly molted crabs were measured immediately after molt. Megalopae from all dietary treatments successfully molted to C1; however, best survival (70%) was achieved by megalopae fed MBD containing a fish oil/corn oil ratio of 1:1. Megalopae fed MBD containing ratios of 3:1and 1:0 showed survival of 65%, while survival of megalopae fed either live Artemia nauplii or MBD containing a fish oil/corn oil ratio of 2:1 was 60%. Lower survival (55% and 35%) was recorded for megalopae fed MBD with ratios of 1:2 and 0:1, respectively. Significantly greater mean carapace width (3.51 ± 0.03 mm) and significantly higher mean dry weight (2.14 ± 0.14 mg) was recorded for crabs molting from megalopae fed live Artemia nauplii compared to those resulting from megalopae fed MBD. Results from this experiment indicate that the optimal fish oil/corn oil ratio is around 1:1 when oil is supplied at a level of 6% of total diet dry weight. The study further showed that complete replacement of fish oil with corn oil in the formulated diet resulted in a high occurrence of MDS-related mortality, indicating an essential dietary requirement for >C18 highly unsaturated fatty acids (HUFA), and a link between n-3 HUFA availability and the occurrence of MDS. In nature the nutritional requirements of crab larvae are satisfied by the diversity and variability of natural prey and their broad nutrient contents. As the study of larvae in the wild is practically impossible, analysis of the biochemical changes occurring in the tissue during larval development in the laboratory has been used as an alternative method for elucidating information on the nutritional requirements of larvae. These principles have been applied successfully to studies of several crustaceans, but a complete record of changes in lipid and fatty acid profile during ontogenetic development of mud crabs was not available. Comprehensive analysis of changes in dry weight, organic content, total lipid, lipid class and fatty acid composition in developing S. serrata larvae reared under standard hatchery conditions is reported in Chapter 7. An increase in mean dry weight (DW) was observed during larval development, from 12.11 ± 0.31 μg for newly hatched zoea I, to 1025.52 ± 87.11 μg for newly molted megalopae.

The organic content of larvae ranged between 58.18 ± 2.37% (zoea I) and 70.16 ± 0.68% (zoea III) of sample DW, and statistical analysis indicated significant differences in the percentage organic content between the larval stages. On this basis the results relating to fatty acid composition of larvae were presented on a per unit ash free dry weight (AFDW) basis. The total fatty acid content of larval tissues increased from 22.89 ± 3.72 μg mg-1 AFDW in newly hatched zoea I larvae, peaking at 31.38 ± 18.30 μg mg-1 AFDW at zoea V before dropping down to 24.92 ± 13.56 μg mg-1 AFDW at the megalopa stage. During the first zoea stage, saturated fatty acids (SFA) and highly unsaturated fatty acids (HUFA), dominated the fatty acid profile, while monounsaturated fatty acids (MUFA) were less abundant. As the larval developed the HUFA were depleted, while an increase in MUFA was recorded during the zoea V and megalopa stages, suggesting that HUFA requirements go down during the later part of larval development. The fatty acid composition was relatively stable throughout larval development, dominated by palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1n- 9), arachidonic acid (20:4-n-6, AA), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA). A substantial increase in linolenic acid (18:3n-3, LNA) levels was recorded in zoea V larvae, a result probably linked to the elevated LNA content in enriched Artemia meta-nauplii used as food for later stage larvae.

When subjected to starvation, crustaceans go through distinct phases of biomass degradation. This process is useful for identification of key fatty acids, and Chapter 8 reports on the changes in lipid and fatty acid metabolism observed in zoea V and megalopa subjected to feeding and starvation, respectively. The objective was to gain a better understanding of fatty acid utilization, and to identify possible changes in lipid requirements during the two last larval stages, which again may be linked to the high incidence of MDS during these larvae stages in mud crab hatcheries. Larvae were reared following established hatchery protocols, and newly molted zoea V and megalopae were subject to 4 days of feeding or starvation, and larval tissue was sampled for lipid analysis. An additional 6 day starvation treatment was incorporated for the megalopa stage as a response to the longer duration of this larval stage compared to the zoea stages. The results showed that fatty acids had an important role as an energy store in starved larvae, and the fatty acid content of larval tissue was reduced by 71.93% and 72.96% for zoea V and megalopa, respectively, during the 4 day starvation period.

Further depletion of stored fatty acids was not, however, observed when megalopae were starved for another 2 days, indicating that other nutrient sources are utilized for energy when larvae are subject to prolonged starvation. Fatty acids from the polar lipid fraction dominated larval tissue, while fatty acids from the neutral lipid fraction were preferentially metabolized for energy during food deprivation. Depletion of both SFA, MUFA and HUFA was observed among unfed larvae, and in accordance with the findings in the previous chapter, depletion of EPA, DHA and AA indicated a possible decreasing requirement of these fatty acids during later larval development. Fed larvae, on the other hand, maintained more stable fatty acid levels, and they were able to sequester a range of fatty acids from live Artemia. Comparison of the lipid profile of starved and fed larvae highlights an inability for de novo synthesis of both C18 fatty acids such as LNA, and HUFA like DHA, EPA and AA.

This project utilised integrated methodology to study lipid requirements of mud crab larvae. Growth trials were the most optimal way of specifying nutritional requirement, while biochemical analysis was used to gain a deeper understanding of the underlying mechanisms behind growth promotion. The findings have significantly enhanced our current understanding of dietary lipid requirements and utilization in mud crab larvae, which will have significant implications for the development of a nutritionally appropriate MBD for S. serrata larval culture in the future.

Item ID: 2150
Item Type: Thesis (PhD)
Keywords: Scylla serrata, mud crabs, larvae, megalopa stage, aquaculture, hatcheries, lipid nutrition, microbound diet
Date Deposited: 09 Apr 2009 04:02
FoR Codes: 07 AGRICULTURAL AND VETERINARY SCIENCES > 0702 Animal Production > 070204 Animal Nutrition @ 0%
06 BIOLOGICAL SCIENCES > 0606 Physiology > 060603 Animal Physiology Systems @ 0%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070401 Aquaculture @ 0%
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