Influence of Moult Cycles on Digestive Enzyme Activities during Early Larval Stages of Panulirus ornatus

The tropical spiny lobster, Panulirus ornatus, has a complex life cycle characterised by a series of moults that occur throughout pelagic larval stages. Significant morphological, physiological, and biochemical changes commonly coincide with moulting and can have dietary implications when culturing this species. Digestive enzyme activities respond to nutritional requirements and have provided useful insight into nutrient use dynamics associated with first-feeding in P. ornatus. Beyond first-feeding, however, information on digestive enzyme activities in P. ornatus is scarce. Greater knowledge of fluctuations in digestive enzyme activities during moult cycles should facilitate better formulation of feeds and more efficient feeding regimens. As an initial step towards this goal, the present study evaluated the influence of moult cycles on digestive enzyme activities during early larval stages of P. ornatus. The investigation focused exclusively on early larval stages (stages I-III) when delivery of appropriate feeds and nutrition can dramatically affect subsequent growth and survival

Development of a formulated diet for mud crab, Scylla sewata, larvae

This study aimed to develop a suitable formulated diet to replace live food in mud crab, Scylla serrata, larval culture. A microbound diet (MBD) was formulated based on known requirements of other crustaceans and was assessed for ingestion and retention by the various larval stages of S. serrata. Ingestion and retention was determined by labeling the MBD with ¹⁴C, and subsequently measuring ¹⁴C in larvae fed these diets. After it was shown that the larvae readily accepted MBD, different types of binders were tested to determine those best suited for MBD prepared for S. serrata larvae. The diet particle size preference and optimal feeding ration were then determined for the various larval stages. Finally, the diet was tested in combination with different ratios of Artemia as a food source for Megalopa. \ud \ud Fundamental to the success of the study was the development of methods that would ensure routine and reliable production of mud crab larvae. Larval production runs were tried using protocols from various authors and procedures that showed positive results after several trials were adopted. A progressive improvement in survival was achieved towards the end of the study and the rearing protocol that was finally adopted has now formed the basis for hatchery production of S. serrata at James Cook University. A technique for evaluating fish larvae using dietary ¹⁴C was refined and adopted in this study for the measurement of ingestion and retention of the MBD by S. serrata larvae. Several studies were cond ucted to serve as basis to refining this technique. Based on an experiment to determine factors that could affect the measurement of the ¹⁴C content of larvae fed ¹⁴ C labelled MBD, it was found that S. serrata larvae do not absorb the ¹⁴C that leaches from the diet but MBD particles that stick to the larvae were the major source of potential error. As such, it was found necessary to include a control treatment with dead larvae when running an experiment so that the radioactivity reading of the dead larvae can be used to correct the radioactivity readings in the treatments when measuring ingestion. \ud \ud Newly hatched S. serrata zoea readily ingested the MBD and ingestion increases with larval age. Ingestion of MBD did not vary significantly between Zoea I and Zoea II and also between Zoea III and Zoea IV; ingestion by other larval stages were significantly different from each other. Ingestion of MBD by Megalopa was found comparable to previously reported ingestion of live food (8 Artemia larva⁻¹ h⁻¹). Studies were also conducted to determine the duration of diet exposure that resulted in maximal ingestion of the MBD. Results showed that for Zoea I to Zoea III and Megalopa, feeding for 1 h resulted in maximal ingestion as there was no further increase in ingestion with longer diet exposure. For Zoea IV and Zoea V, at least 2 h was required for maximal ingestion as there was no further increase in ingestion after 2 h. The gut residence times (GRT) of MBD for the various larval stages of S. serrata were determined in order to know the required period that should be allowed for the larvae to empty their gut when measuring retention of the MBD. In Zoea I, GRT was found to be 1 h as retention of the MBD significantly decreased after 1 h following removal of available MBD. In Zoea III and Zoea IV, GRT was found to be 2 h; retention of the MBD significantly decreased after 2 h following food removal and there was no further significant decrease in retention after this time. Results did not clearly indicate the GRT of Zoea V and Megalopa, but there were indications suggesting longer GRT of around 4- 5 h for these stages. \ud \ud Studies to test the suitability of different binders (agar, alginate, carrageenan, gelatin and zein) for MBD showed that there were no significant differences in ingestion and retention of MBD resulting from binder type. Further evaluation of these binders, based on leaching of radioactivity from diets, showed that least leaching was found in zeinbound MBD. Since the greater leaching of nutrients from die ts with other types of binders did not make these diets more attractive (i.e. result in significantly greater rates of ingestion), the minimal leaching of nutrients from zein-bound MBD made zein the more desirable binder. Unnecessary leaching wastes important dietary components and can result in deterioration of water quality. \ud \ud The particle size preference and optimal feed ration were determined for the various larval stages of S. serrata based on larval ingestion of ¹⁴C labeled MBD. The results provided important information for feeding management of S. serrata and allowed recommendation of the most suitable MBD particle size range and ration for each of the larval stages of S. serrata (i.e. for Zoea I, <150 μm MBD particles given at 5.4 mg L⁻¹; for Zoae III, 150-250 μm MBD particles fed at 7.1 mg L⁻¹; for Zoea V, 250-400 μm MBD particles fed at 8.2 mg L⁻¹; for Megalopa, 400-600 μm MBD particles given at 2 mg L⁻¹). It was found that the optimal particle size ranges for different larval stages are not completely provided by a rotifer/Artemia diet commonly used in mud crab hatcheries. This highlights the advantage of using MBD since they can be prepared within any desired particle size range and as such offer the potential to provide a more appropriate diet to S. serrata larvae. The results also suggested that MBD, provided at a rate equivalent to 50% of the dry weight of the 'standard' live food diet is the optimal ration for Zoea I to Zoea V larvae and it could be as low as 12.5 % for Megalopa. The potentia l for complete and partial replacement of Artemia with MBD for Megalopa was also tested. Survival of megalopae to crab stage did not vary significantly between the different ratios of MBD and Artemia, but a combination of 25 % MBD and 75% Artemia consistently gave the highest survival. Treatments receiving high proportions of MBD molted earlier compared to those receiving high proportions of Artemia. In another experiment where Megalopa were reared individually, 90 % survival to crab stage was achieved in both treatments fed either MBD or Artemia only. The megalopae fed MBD only also molted one day earlier than those fed Artemia only. These results showed that the MBD was capable of supporting successful molting of megalopae to crab stage and the possibility of complete replacement of Artemia with MBD. It was also shown that while both the MBD and the Artemia were adequate feeds on their own, a combination of the two in an appropriate proportion may give improved results

Digestive enzyme dynamics during early life stages of the mud crab, Scylla serrata and the spiny lobster, Panulirus ornatus

The mud crab, Scylla serrata and the spiny lobster, Panulirus ornatus, are high value crustaceans in the tropics and sub-tropics of the Indo-Pacific region subject to intense fisheries pressure, particularly in Asia where no catch limits are imposed and fishery laws are often not strictly enforced. Because of increasing demand and dwindling fisheries landings, interest in aquaculture of both species has grown strongly over recent years. However, continued dependence on wild seed for stocking has been a major bottleneck for expansion and further development of aquaculture industries for both species. Hatchery techniques for these species have received significant research attention over recent years but considerable development is required to further improve survival and bring commercial viability to hatchery operations. One of the most important yet poorly understood components of hatchery production of crustaceans is larval nutrition, particularly the aspects of larval nutritional requirements and digestive capacity. Larvae of aquatic animals, particularly early larvae, rely primarily on chemical digestion of ingested foods with the aid of enzymes. This study assayed the major digestive enzymes during larval development of both S. serrata and P. ornatus to assess larval capacity to digest major nutrients and to evaluate the relative utilization of these nutrients for energy: (a) during embryonic development and starvation of the newly hatched larvae (Chapter 3); (b) under different conditions of intermittent food availability (Chapter 4); (c) in response to different food quantity and quality (Chapter 5); and (d) at different stages of the moult cycle and larval ontogeny (Chapter 6). Following the general introduction (Chapter 1) and general materials and methods (Chapter 2) sections, changes in activities of the three major digestive enzymes (amylase, protease and esterase) during embryonic development of S. serrata and P. ornatus, as well as in unfed newly hatched larvae, were examined in Chapter 3. For both species, esterase activities started to increase significantly during the early phase of embryonic development while amylase and protease activities remained at about the same levels, suggesting that lipids were the nutrients most heavily utilized during the early embryonic development in both species. However, towards the end of embryonic development, amylase and protease activities increased while esterase activities showed decreasing trends, suggesting that as lipid reserves were depleted and became insufficient to meet the increasing energy demand, protein reserves, and to some extent carbohydrates, were increasingly utilized. It was further shown that proteins continued to be the main energy source of newly hatched larvae during the initial phase of starvation for both S. serrata and P. ornatus as higher levels of protease compared to esterase and amylase were present in starved newly hatched larvae of both species. Chapter 4 was designed to examine changes in the major digestive enzyme activities of first feeding larvae of S. serrata and P. ornatus subject to different food availability conditions: (a) when food was immediately available (fed) vs. when food was not immediately available (starved); (b) when food was initially available (fed) and then withdrawn; and (c) when initial feeding was delayed for different durations. These experiments were intended to obtain insights into how first feeding larvae manipulate their enzyme activities in order to adapt to various conditions of intermittent food availability likely to occur in their natural environment, which should provide useful information for the development of larval formulated diets and hatchery feeding protocols. The enzyme activity responses of first feeding zoeae of S. serrata suggested that protein reserves were the main energy source while no food was available, but where food is available, first feeding zoeae spared proteins and utilized carbohydrates and lipids more extensively. In starved zoeae, protease activity, which was comparably much higher than amylase and esterase activities, remained high throughout the 72 h sampling duration. In contrast, protease activity in fed zoeae initially decreased sharply to very low levels although it eventually increased prior to moulting. Meanwhile, amylase and esterase activities gradually increased, suggesting that fed larvae were possibly building-up protein reserves. The enzyme reponses of first feeding P. ornatus phyllosoma suggest their ability to utilize all three major nutrients, i.e., carbohydrates, proteins and lipids, but also highlight their capacity to prioritise the use of carbohydrates when fed. This was illustrated by the immediate and notable increase in amylase activity in fed phyllosoma, which remained high over following days, however such a phenomenon was not observed in starved larvae. When food was removed after the phyllosoma were fed for 24 h, amylase activity decreased back to low levels, suggesting that the phyllosoma quickly responsed to the withdrawal of food by substantially reducing their utilization of carbohydrates and shifting to greater utilization of proteins and lipids. Newly hatched zoeae of S. serrata and phyllosoma of P. ornatus both showed an ability to compensate for delayed food availability by increasing amylase activity when initial feeding was delayed for varying durations. Both specific and total amylase activities of newly hatched zoeae where feeding was delayed for a period of 12 h to 36 h, were significantly higher than those of larvae fed immediately after hatching for the same 12 h duration. In newly hatched phyllosoma where feeding was delayed for a period of 24 h before being fed for 24 h, both specific and total amylase activities were also significantly higher than those fed immediately after hatching for the same 24 h period. However, such a response was no longer observed when feeding was delayed beyond 24 h, suggesting that the ability of newly hatched phyllosoma to compensate for the delayed food intake diminished as the starvation period extended. In Chapter 5, the digestive enzyme responses of larvae to quantity and quality of foods, particularly in terms of food density and food type were investigated. The results of this chapter clearly showed that both food quantity and quality significantly influenced larval digestive enzyme dynamics. Both S. serrata and P. ornatus larvae showed an ability to maximize the utilization of available food by increasing their digestive enzyme activities in response to increasing prey density. Comparison of enzyme activities of Zoea I S. serrata fed different densities of rotifers showed that the rotifer densities that resulted in maximal digestive enzyme activities fell within the range considered optimal for larval rearing of this species. A similar result was obtained for Stage I phyllosoma of P. ornatus fed different densities of Artemia nauplii. These results together suggest that digestive enzyme activity can be a good indicator of appropriate prey density used in larval rearing. The digestive enzyme responses of Zoea II and megalopae of S. serrata to different types of food helped identify their relative nutritional values to the larvae. The digestive enzyme responses of Zoea II fed rotifers and Artemia, respectively, largely reflected the relative proximate contents of these two most commonly used hatchery prey. Similarly, the enzyme activities of megalopae fed either a microbound diet (MBD) developed in this laboratory or Artemia also indicated differences in digestibility and nutritional quality of Artemia compared to the formulated MBD. Huge differences in amylase activity detected between megalopae fed the MBD and those fed Artemia indicated a possible significant role of dietary carbohydrates in megalopal nutrition and a carbohydrate deficiency in the MBD used. In Chapter 6, changes in digestive enzyme activities during the moult cycle and in the course of larval ontogeny of S. serrata and P. ornatus were examined. The results of activity changes in the major digestive enzymes related to the moult cycle of newly hatched Zoea I and Megalopa as the postlarvae of S. serrata, as well as Stage I and II phyllosoma of P. ornatus, provided insights into the utilization of major nutrients during the recurring episodes of feeding activity changes related to the moult cycle of larvae. During the moult cycle of S. serrata Zoea I, newly hatched zoeae appeared to initially spare proteins and relied more on carbohydrates and lipids for energy, but as the moult cycle progressed, all three major nutrients were utilized when the larvae were actively feeding during the inter-moult stage. On the other hand, newly moulted megalopae seemed to initially utilize carbohydrates and proteins heavily while sparing lipids, however, as the moult cycle advanced, all three major nutrients were utilized and, during the second half of the moult cycle, megalopae relied more on lipids. During the moult cycle of P. ornatus Stage I phyllosoma, newly hatched larvae initially utilized carbohydrates and proteins to a greater extent, however as they developed towards moulting, there was a trend of gradually increasing lipid utilization. During the initial phase of the moult cycle of Stage II phyllosoma, utilization of carbohydrates and lipids was increased while protein utilization was slightly reduced. During the second half of the moult cycle, lipids were increasingly utilized as the phyllosoma approached moulting. Changes in major digestive enzyme activities as larvae developed progressively into subsequent stages revealed ontogenetic improvements in the digestive capacity of both S. serrata and P. ornatus larvae. Comparison of results of specific and total activities showed that total activity appeared to be a better way of expressing changes in enzyme activities during larval ontogeny. In S. serrata larvae, the total activities of the major digestive enzymes clearly increased with larval development but more dramatic improvements occurred at Zoea IV, Zoea V and Megalopal stages. During the development of P. ornatus phyllosoma from Stages I to V, the activities of all three major digestive enzymes also increased from one stage to next stage, but these increases were much more pronounced at Stage IV and V, where protease and esterase activities more than doubled. These results suggest that significant improvements in digestive capacity occur from Zoea IV onward for S. serrata and from Stage IV for P. ornatus, which may imply better chances of success in introducing formulated diets at, or beyond, these larval stages. Chapter 7 summarizes the results of this study and offers general discussion within a broader context. Overall, the results of this research clearly demonstrated that analysis of digestive enzyme activities is a valuable method for the study of larval nutrition. For example, digestive enzyme activities could be used to indicate the relative utilization of various nutrients by the developing embryo and newly hatched larvae, providing useful information that could be used in the formulation of broodstock diets that could improve the quality of the newly hatched larvae. The enzyme responses of larvae to various conditions of food availability, food quality and quantity, as well as during recurring moulting cycles, also provides important information that could be used as the basis for devising appropriate larval feeding regimes and feeding protocols in hatcheries. Furthermore, changes in enzyme activities during larval ontogeny provide information on the digestive capacity of the various larval stages, providing clues regarding the suitable timing for introduction of formulated diets

Digestive enzyme dynamics during early life stages of the mud crab, Scylla serrata and the spiny lobster, Panulirus ornatus

The mud crab, Scylla serrata and the spiny lobster, Panulirus ornatus, are high value crustaceans in the tropics and sub-tropics of the Indo-Pacific region subject to intense fisheries pressure, particularly in Asia where no catch limits are imposed and fishery laws are often not strictly enforced. Because of increasing demand and dwindling fisheries landings, interest in aquaculture of both species has grown strongly over recent years. However, continued dependence on wild seed for stocking has been a major bottleneck for expansion and further development of aquaculture industries for both species. Hatchery techniques for these species have received significant research attention over recent years but considerable development is required to further improve survival and bring commercial viability to hatchery operations. One of the most important yet poorly understood components of hatchery production of crustaceans is larval nutrition, particularly the aspects of larval nutritional requirements and digestive capacity. Larvae of aquatic animals, particularly early larvae, rely primarily on chemical digestion of ingested foods with the aid of enzymes. This study assayed the major digestive enzymes during larval development of both S. serrata and P. ornatus to assess larval capacity to digest major nutrients and to evaluate the relative utilization of these nutrients for energy: (a) during embryonic development and starvation of the newly hatched larvae (Chapter 3); (b) under different conditions of intermittent food availability (Chapter 4); (c) in response to different food quantity and quality (Chapter 5); and (d) at different stages of the moult cycle and larval ontogeny (Chapter 6). Following the general introduction (Chapter 1) and general materials and methods (Chapter 2) sections, changes in activities of the three major digestive enzymes (amylase, protease and esterase) during embryonic development of S. serrata and P. ornatus, as well as in unfed newly hatched larvae, were examined in Chapter 3. For both species, esterase activities started to increase significantly during the early phase of embryonic development while amylase and protease activities remained at about the same levels, suggesting that lipids were the nutrients most heavily utilized during the early embryonic development in both species. However, towards the end of embryonic development, amylase and protease activities increased while esterase activities showed decreasing trends, suggesting that as lipid reserves were depleted and became insufficient to meet the increasing energy demand, protein reserves, and to some extent carbohydrates, were increasingly utilized. It was further shown that proteins continued to be the main energy source of newly hatched larvae during the initial phase of starvation for both S. serrata and P. ornatus as higher levels of protease compared to esterase and amylase were present in starved newly hatched larvae of both species. Chapter 4 was designed to examine changes in the major digestive enzyme activities of first feeding larvae of S. serrata and P. ornatus subject to different food availability conditions: (a) when food was immediately available (fed) vs. when food was not immediately available (starved); (b) when food was initially available (fed) and then withdrawn; and (c) when initial feeding was delayed for different durations. These experiments were intended to obtain insights into how first feeding larvae manipulate their enzyme activities in order to adapt to various conditions of intermittent food availability likely to occur in their natural environment, which should provide useful information for the development of larval formulated diets and hatchery feeding protocols. The enzyme activity responses of first feeding zoeae of S. serrata suggested that protein reserves were the main energy source while no food was available, but where food is available, first feeding zoeae spared proteins and utilized carbohydrates and lipids more extensively. In starved zoeae, protease activity, which was comparably much higher than amylase and esterase activities, remained high throughout the 72 h sampling duration. In contrast, protease activity in fed zoeae initially decreased sharply to very low levels although it eventually increased prior to moulting. Meanwhile, amylase and esterase activities gradually increased, suggesting that fed larvae were possibly building-up protein reserves. The enzyme reponses of first feeding P. ornatus phyllosoma suggest their ability to utilize all three major nutrients, i.e., carbohydrates, proteins and lipids, but also highlight their capacity to prioritise the use of carbohydrates when fed. This was illustrated by the immediate and notable increase in amylase activity in fed phyllosoma, which remained high over following days, however such a phenomenon was not observed in starved larvae. When food was removed after the phyllosoma were fed for 24 h, amylase activity decreased back to low levels, suggesting that the phyllosoma quickly responsed to the withdrawal of food by substantially reducing their utilization of carbohydrates and shifting to greater utilization of proteins and lipids. Newly hatched zoeae of S. serrata and phyllosoma of P. ornatus both showed an ability to compensate for delayed food availability by increasing amylase activity when initial feeding was delayed for varying durations. Both specific and total amylase activities of newly hatched zoeae where feeding was delayed for a period of 12 h to 36 h, were significantly higher than those of larvae fed immediately after hatching for the same 12 h duration. In newly hatched phyllosoma where feeding was delayed for a period of 24 h before being fed for 24 h, both specific and total amylase activities were also significantly higher than those fed immediately after hatching for the same 24 h period. However, such a response was no longer observed when feeding was delayed beyond 24 h, suggesting that the ability of newly hatched phyllosoma to compensate for the delayed food intake diminished as the starvation period extended. In Chapter 5, the digestive enzyme responses of larvae to quantity and quality of foods, particularly in terms of food density and food type were investigated. The results of this chapter clearly showed that both food quantity and quality significantly influenced larval digestive enzyme dynamics. Both S. serrata and P. ornatus larvae showed an ability to maximize the utilization of available food by increasing their digestive enzyme activities in response to increasing prey density. Comparison of enzyme activities of Zoea I S. serrata fed different densities of rotifers showed that the rotifer densities that resulted in maximal digestive enzyme activities fell within the range considered optimal for larval rearing of this species. A similar result was obtained for Stage I phyllosoma of P. ornatus fed different densities of Artemia nauplii. These results together suggest that digestive enzyme activity can be a good indicator of appropriate prey density used in larval rearing. The digestive enzyme responses of Zoea II and megalopae of S. serrata to different types of food helped identify their relative nutritional values to the larvae. The digestive enzyme responses of Zoea II fed rotifers and Artemia, respectively, largely reflected the relative proximate contents of these two most commonly used hatchery prey. Similarly, the enzyme activities of megalopae fed either a microbound diet (MBD) developed in this laboratory or Artemia also indicated differences in digestibility and nutritional quality of Artemia compared to the formulated MBD. Huge differences in amylase activity detected between megalopae fed the MBD and those fed Artemia indicated a possible significant role of dietary carbohydrates in megalopal nutrition and a carbohydrate deficiency in the MBD used. In Chapter 6, changes in digestive enzyme activities during the moult cycle and in the course of larval ontogeny of S. serrata and P. ornatus were examined. The results of activity changes in the major digestive enzymes related to the moult cycle of newly hatched Zoea I and Megalopa as the postlarvae of S. serrata, as well as Stage I and II phyllosoma of P. ornatus, provided insights into the utilization of major nutrients during the recurring episodes of feeding activity changes related to the moult cycle of larvae. During the moult cycle of S. serrata Zoea I, newly hatched zoeae appeared to initially spare proteins and relied more on carbohydrates and lipids for energy, but as the moult cycle progressed, all three major nutrients were utilized when the larvae were actively feeding during the inter-moult stage. On the other hand, newly moulted megalopae seemed to initially utilize carbohydrates and proteins heavily while sparing lipids, however, as the moult cycle advanced, all three major nutrients were utilized and, during the second half of the moult cycle, megalopae relied more on lipids. During the moult cycle of P. ornatus Stage I phyllosoma, newly hatched larvae initially utilized carbohydrates and proteins to a greater extent, however as they developed towards moulting, there was a trend of gradually increasing lipid utilization. During the initial phase of the moult cycle of Stage II phyllosoma, utilization of carbohydrates and lipids was increased while protein utilization was slightly reduced. During the second half of the moult cycle, lipids were increasingly utilized as the phyllosoma approached moulting. Changes in major digestive enzyme activities as larvae developed progressively into subsequent stages revealed ontogenetic improvements in the digestive capacity of both S. serrata and P. ornatus larvae. Comparison of results of specific and total activities showed that total activity appeared to be a better way of expressing changes in enzyme activities during larval ontogeny. In S. serrata larvae, the total activities of the major digestive enzymes clearly increased with larval development but more dramatic improvements occurred at Zoea IV, Zoea V and Megalopal stages. During the development of P. ornatus phyllosoma from Stages I to V, the activities of all three major digestive enzymes also increased from one stage to next stage, but these increases were much more pronounced at Stage IV and V, where protease and esterase activities more than doubled. These results suggest that significant improvements in digestive capacity occur from Zoea IV onward for S. serrata and from Stage IV for P. ornatus, which may imply better chances of success in introducing formulated diets at, or beyond, these larval stages. Chapter 7 summarizes the results of this study and offers general discussion within a broader context. Overall, the results of this research clearly demonstrated that analysis of digestive enzyme activities is a valuable method for the study of larval nutrition. For example, digestive enzyme activities could be used to indicate the relative utilization of various nutrients by the developing embryo and newly hatched larvae, providing useful information that could be used in the formulation of broodstock diets that could improve the quality of the newly hatched larvae. The enzyme responses of larvae to various conditions of food availability, food quality and quantity, as well as during recurring moulting cycles, also provides important information that could be used as the basis for devising appropriate larval feeding regimes and feeding protocols in hatcheries. Furthermore, changes in enzyme activities during larval ontogeny provide information on the digestive capacity of the various larval stages, providing clues regarding the suitable timing for introduction of formulated diets

Management strategies for grow-out culture of mud crab

There is an increasing interest in mud crab farming because of the growing demand for mud crab in domestic and international markets. Different methods for rearing crabs in ponds, pens and cages have evolved through several years of research and experiences of farmers. Mud crabs are cultured in brackishwater earthen ponds and pens in mangroves. Fattening of lean crabs is also integrated with the grow-out culture system. Cannibalism is one of the major factors affecting the survival of crabs in growout ponds and pens. Hence, various strategies are recommended to reduce cannibalism such as stocking density of less than 2,000 crabs ha-1, provision of suitable shelters, sufficient quantity of natural food and formulated feeds that are evenly distributed in the pond or pen. Feeding rate used in the pen is adjusted to avoid excess feeds that can attract rats and other land animals that can damage the enclosures. Selective harvesting is normally practiced since mud crabs do not grow or get fattened at the same time even if they belong to the same batch

Evaluation of earthworm meal (<em>Eudrilus eugeniae</em>) as fish meal replacement in juvenile Mangrove Crab (<em>Scylla serrata</em>) diet

Two feeding trials were conducted to evaluate the performance of the earthworm (Eudrilus eugeniae) meal (vermimeal) as a replacement for fish meal in the diet of juvenile mangrove crab (Scylla serrata) in terms of growth, feed efficiency, intermolt duration, carapace width increment, molting success, survival, and incidence of cannibalism. Four experimental diets were formulated with 0, 25, 50, and 75% fish meal protein replaced with earthworm meal protein, designated as D0, D25, D50, and D75, respectively. In the first feeding trial, the experimental diets were fed to crablets with an initial average weight of 0.09±0.007g. Each replicate comprised ten (10) crablets individually stocked in 750ml-capacity plastic containers randomly distributed in a static water bath. Final weight gain, feed efficiency and survival were measured after the 5^th^ molt. In the second feeding trial, 150 crablets with an average weight of 0.07±0.002g were stocked at 30 crablets each in five (5) replicate tanks. Survival and incidence of cannibalism were monitored daily until the end of the culture period of 30 days. The first study showed that the growth of mangrove crab juveniles fed the diet with up to 50% of the fish meal protein replaced by the earthworm meal protein was statistically comparable to the control group. The survival of crablets in the second feeding trial was highest in D25 (33%), followed by D50 (29%), D0 (18%), and lowest in the group fed with D75 (13%). Overall, earthworm meal (Eudrilus eugeniae) can replace as much as 50% of the fish meal protein in juvenile mangrove crab (Scylla serrata) diet without negative effects on growth, carapace width increment, feed efficiency, intermolt duration, molting success, survival, and cannibalism

Diet particle size preference and optimal ration for mud crab, Scylla serrata, larvae fed microbound diets

This paper reports on experiments to determine particle size preference and optimal ration for the various larval stages of the mud crab, Scylla serrata, fed microbound diets (MBD). All experiments used 14C-labelled rotifers as components of MBD, and ingestion was determined by assessing the 14C content of S. serrata larvae after feeding on the MBD. Five size ranges of MBD (<150, 150–250, 250–400, 400–600 and 600–800 μm) were used to assess ingestion by Zoea I, Zoea III, Zoea V and Megalopa. All were fed a ration equivalent to twice the equivalent dry weight of live foods provided to each larval stage under standard rearing conditions (standard ration). MBD particle size preference increased with increasing larval development. Highest ingestion rates by Zoea I, Zoea III, Zoea V and Megalopa were found for particle size ranges of <150, 150–250, 250–400 and 400–600 μm, respectively. MBD within these particle size ranges were used in subsequent experiments to assessed optimum rations for each of the four larval stages. Larvae were fed rations based on multiples or fractions of the standard ration (100%): 12.5%, 25%, 50%, 100%, 200% and 300%. There were no significant increases in ingestion, for any of the four larval stages, when ration was increased above 100%. For Zoea I, Zoea III and Zoea V larvae, there was no significant difference in the rate of MBD ingestion when ration was reduced to 50%; however, a 25% ration brought about a significant decrease in ingestion. There was no significant decrease in the rate of ingestion by Megalopa when ration was reduced from 100% to either 50%, 25% or 12.5%. The results suggest that rations currently used for larval rearing of S. serrata may be excessive. Little is currently known of the feeding behaviour and nutritional requirements of S. serrata larvae, and the results of this study are a significant development in this field

Grow-out of the tiger shrimp Penaeus monodon in floating net cages in Batan Bay, northern Panay

The effects of two stocking densities and two feed combinations on growth, survival and production of the giant tiger shrimp Penaeus monodon in floating net cages were studied in an attempt to refine the existing technology on cage culture of tiger shrimp for the benefit of small- and medium-scale fish farmers. Four treatments were tested, replicated in time: stocking density of 100 shrimp/m2, feeding with 70% commercial shrimp pellets P and 30% 'trash fish' F; 100 shrimp/m2 and 50% P + 50% F; 200 shrimp/m2 and 70% P + 30% F; and 200 shrimp/m2 and 50% P + 50% F. Harvested after 93–95 d, the shrimps at the lower density treatments were significantly larger and had greater proportion of good sizes (>18 g body weight). Although the combination of 70% P + 30% F resulted in better growth, the combination of 50% P + 50% F resulted in more good-size shrimps. Survival, production, and gross income were not significantly different among treatments. None of the treatments in this study was economically viable. Gross income was very low due to poor growth and survival, mostly due to luminous vibriosis, then a new disease that eventually wiped out many shrimp hatcheries and ponds around Panay Island

Determining ingestion of microbound diet particles by mud crab, Scylla serrata, larvae

This study evaluated 14C-labelling as a tool to investigate the suitability of microbound diet (MBD) particles for the various larval stages of the mud crab, Scylla serrata. The results showed that S. serrata larvae held in seawater containing 14C leachate from 14C-labelled MBD particles (after MBD particles had been removed) did not contain levels of 14C above background. However, dead S. serrata larvae held in water with 14C-labelled MBD particles had a significantly higher 14C content than background even after thorough washing. They suggested that MBD particles trapped on the body of crab larvae cannot be completely washed off, and they represent a source of error that needs to be corrected when using this technique to determine ingestion of MBD particles by crab larvae. Consequently, a control treatment consisting of dead larvae held with 14C-labelled MBD particles was used to correct larval ingestion data in subsequent experiments. To determine larval ingestion of MBD particles, each larval stage of S. serrata was fed 14C-labelled MBD over a five-hour period and their radioactive contents were determined hourly. MBD particles were readily ingested by all larval stages of S. serrata. Ingestion of MBD particles increased with larval development with megalopa consuming more than three times the amount of MBD than Zoea V and more than ten times that of Zoea IV larvae. Ingestion of MBD particles by Zoea I declined after four hours, whereas ingestion by Zoea II, III and megalopa did not differ significantly over the five-hour feeding period. Zoea IV and V showed greater levels of MBD ingestion after four and five hours than after one hour. The present results suggest that the time to reach maximal ingestion, as indicated by the time to achieve maximum larval 14C content, for Zoea I, II, III and megalopa is one hour, but is greater for Zoea IV and V. The results of this study allow fine-tuning of a technique with great potential for investigating aspects of the nutrition of S. serrata larvae and indicate clear potential for developing successful MBD for them

Preliminary assessment of a microbound diet as an Artemia replacement for mud crab, Scylla serrata, megalopa

As an important step toward development of a formulated diet for hatchery culture of the mud crab, Scylla serrata, this paper reports on laboratory experiments to assess the potential of a microbound diet (MBD) as a replacement for Artemia nauplii fed to megalopal larvae of S. serrata. The effects of different proportions of dietary MBD and Artemia on survival and moulting success of megalopa to the crab stage were investigated. In the first experiment, megalopae were reared communally and fed either 100% MBD, 100% Artemia or different combinations of the two (75%:25%, 50%:50%, 25%:75%). The experiment was terminated when all larvae had either metamorphosed or died. Larvae fed a combination of 25% MBD and 75% Artemia consistently showed the highest survival among all treatments throughout the experiment. Survival of larvae fed 100% MBD was the lowest early in the experiment but improved to become the second highest toward the end of the culture period. Overall survival of larvae fed 100% MBD did not differ significantly from that of larvae fed 100% Artemia. Moulting to the crab stage began on day 7 for larvae in the treatment receiving a 50%:50% combination of MBD and Artemia. On day 8, all larvae in treatments receiving greater than 25% MBD had some first stage crabs. Larvae fed Artemia only were the last to moult to the juvenile crab stage, but moulting occurred simultaneously on day 10. Because of cannibalism observed in the first experiment, a second experiment was conducted where megalopae were reared individually and fed either 100% Artemia or 100% MBD. Ninety percent of larvae from both treatments successfully moulted to the crab stage. Again, megalopae fed MBD began moulting 1 day ahead of those fed Artemia. The results of these studies show that the MBD used contained all necessary nutrients to sustain successful moulting of S. serrata megalopae to the crab stage. The acceptability of MBD by S. serrata larvae suggests significant potential for using the MBD in future experiments to investigate larval nutritional requirements of this commercially important crab species. Indeed, the more rapid moulting of larvae fed MBD in both experiments suggests that the MBD may have contained certain beneficial nutrients that were not provided by Artemia alone. The fact that no significant differences in survival between megalopae fed 100% MBD and those fed 100% Artemia in both communal and individual rearing experiments suggests that total replacement of Artemia with MBD is possible for S. serrata megalopae. This could result in substantial savings in operating costs for S. serrata hatcheries