Effect of Stocking Density on the Yield of Larval Mass Production of Kuruma Prawn. Boon-Keng Lim*1 and Kazutsugu Hirayama*1 (Received July 14, 1992)

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1 Nippon Suisan Gakkaishi 59 (2), (1993) Effect of Stocking Density on the Yield of Larval Mass Production of Kuruma Prawn Boon-Keng Lim*1 and Kazutsugu Hirayama*1 (Received July 14, 1992) The effect of initial stocking density (8-47 thousand indiv./m3) on larval mass production of kuruma prawn was studied in ten trials. The larvae were reared for 24 days from the nauplius stage to the 15-16th day of the postlarval stage. The survival rates ranged from 0.29 to 0.84, and were above 0.5 in eight trials. The daily declining pattern of survival numbers in these eight trials colud be expressed by a logarithmic linear regression throughout the rearing period, thus the popu lation decreased in a constant proportion. By a negative correlation between initial stocking density and survival rate, 24-day rearing obtains a survival rate of more than 0.5 with an initial stocking density of less than 30 thousand indiv./m3. Relationship between initial stocking density and harvested biomass per initially stocked individual (final individual fresh body weight ~survival rate) showed a more significant negative correlation. The maximum yield was estimated as ap proximately 80g/m3 at an initial stocking density of 30 thousand indiv./m3. The actual value of the largest yield among these trials was 87g/m3 at an initial stocking density of 25 thousand indiv./ m3. In Japan, larval rearing for mass production of the kuruma prawn Penaeus japonicus is carried with some success. According to national records for artificial larval production, more than 700 million individuals were produced in 1990.*2 Approximately 70% of these larvae were released into coastal waters with the aim of increasing fishery resources. Technical procedures for mass production of kuruma prawn larvae were establish ed thirty years ago, and many studies have been carried out to improve mass culture techniques.1, 2) At present, the rearing techniques have been developed to a settled level. However, there are still some differences in rearing techniques among culturists. They always perform rearing according to their own experience and convenience.3) Under good environmental condi tions, a constant production with higher feeding efficiency as well as savings in labor are important research subjects. Initial stocking density may be a crucial factor in the process of larval produc tion. Wyban et al.4) and Lanari et al.5) reported the effects of fertilization and stocking density on the performance and growth rate of adult prawns of P. vannamei and P. japonicus in an earth pond. Unfortunately, there are still gaps in our knowledge on the effect of initial stocking density on mass production of the larvae. Initial stocking density for larval production is always arbitrarily decided by the culturist's experience. In the present study, initial stocking density for larval rearing was set up at several levels in tanks of 5.5m3 in water capacity. Survival numbers of the larvae were observed daily, and the declining pattern of larval populations throughout the rearing period was recognized. These larval rearing runs were considered to have no harmful influences from diseases or other environmental factors, except for excess stocking density. The relationship of initial stocking density to yield in a 24-day larval mass culture was determined. From this relationship, the potential yield in a mass larval culture was estimat ed. Larval Production Rearing Conditions Larval rearing of kuruma prawns was conducted at the Nippon Sea Farms Inc. Yuya Laboratory, *1 Graduate School of Marine Science and Engineering, Nagasaki University, Bunkyo, Nagasaki 850, Japan ( è åšw m ŽY ÈŠwŒ È). *2 (National records for larval production, larval obtaining and releasing in Fisheries Agency of Japan Sea Farming Fisheries Association, 1992, pp ).

2 Yamaguchi Prefecture, Japan. The spawners were netted from the sea adjacent to Miyazaki Prefecture, Japan. About 30 spawners were placed in a concrete tank, and were allowed to spawn overnight by slowly raising the ambient water temperature from 18 to 27 Ž. The hatched larvae were transferred into a concrete tank (2 ~2 ~1.5m; maximum water capacity 5.5m3) for rearing. Ten trials with various initial stocking populations (Table 1) were carried out over the course of three years. The routine feeding regimens were as follows: diatoms (Skeletonema costatum and Chaetoceros spp., etc.) which had been cultured separately were given to the protozoeal larvae, brine shrimp nauplii were given in the mysis stage and also in the earlier postlarval stage, while the assorted feed (Nippai Co. Ltd.) was supplied after the 7-8th day of the postlarval stage in all trials. Chemical fertilizers were added to energize the diatoms in the rearing tanks, except for one trial (trial no. 6, see Table 1). Total amounts for each supplied food item and the volume of utilized early period of rearing. When it came to the maximum volume, the rearing water was partially replaced (10-20% per once) with fresh sea water. The water temperature of all trials was controlled at 27 }1 Ž from the nauplius stage to the 1st day of the postlarval stage, and was kept at 23 } 1 Ž until the final day of rearing. The feeding procedures and two phases of water temperature controls were applied from conventional rearing techniques of the Yuya Laboratory. The larval stages are named as: nauplius, protozoea, mysis, and postlarva.6) The duration of larval develop mental stages was usually 1 day at the nauplius stage, 5 days at the protozoeal stage, and 3-4 days at the mysis stage. The larvae metamorphos ed into the 1st day of the postlarval stage 9-10 days after hatching. The larval rearing run was carried out for 24 days, until the 15-16th day of the postlarval stage.table 1. Rearing conditions and results of ten trials for mass production of 24-day rearing of kur Rearing Water Quality Dissolved oxygen, ph, specific gravity, nitrate-n,7) nitrite-n,8) and total ammonia-n water are compiled in Table 1. The larvae under observation were always fed sufficiently. The rearing started at 3m3 water volume. Frest sea water was added without replacing in the (ammonia electrode, Orion Research 95-12) in the rearing water for the trials were determined daily (Table 2). Salinity was calculated from specific gravities using Knudsen's table.* The

3 rearing water was aerated almost in a saturated oxygen condition throughout the rearing period. The ph and salinity in these trials fluctuated between 8.5 to 7.9 and 31 to 34 ppt, respectively. These fluctuations are reported to be within the resistance limit of the larvae.9, 10) The addition of potassium nitrate for diatom growth invariably resulted in a high concentration of nitrate-n during the protozoeal stage. The concentration of total ammonia-n increased as rearing went on. However, the concentrations of these two param eters were also below the concentration of toxic level reported by Mawatari and Hirayama.10) Larval Populations Determination of Larval Populations All the hatched larvae were collected from the spawning tank and placed in a polycarbonate container with 30l of sea water. The container water was stirred thoroughly, then 100ml of the water was taken and diluted to 1l in a beaker. Five replications were done. Tenml aliquots of the 1l beakers of water were picked out and the captured larvae were counted. This operation was repeated ten times, and mean larval density in the beakers was obtained. With this, the larval density of the container was estimated. Then the required number for rearing in each tank was stocked according to volumetrically calculated water volume from the container. During rearing, the survival number of the larvae was observed daily. One liter rearing water was sampled at each of eight locations marked along the rearing tank, and all larvae in the sampled water were counted. Larval density in the tank was calculated from the mean of the sample counts in 1l rearing water. The larval population was calculated volumetrically. At the end of rearing, the larvae were harvested using a collecting net which had previously been settled at the water outlet. Three or four thousand individuals were collected from the harvested larvae and weighed to obtain the individual fresh body weight. The total number was calculated gravimetrically, dividing the total weight of the harvested larvae by the individual fresh body weight. With respect to the accuracy of these determina tion methods, it could be considered that the initial and final populations were observed more ac curately than the ones during rearing. Survival Number of the Larvae Figure 1 shows the daily number of surviving larvae on a logarithmic scale in each trial through out the rearing period. Ordinarily, the survival population either becomes lower or remains unchanged as rearing goes on. However, the daily survival number observed with the above method fluctuated up and down. The accuracy of the volumetric estimating method is affected by rearing water temperature and larval size, as reported by Hardin.11) Volume of aeration and stocked number were also considered to be influential factors. Since the water temperature

4 polycarbonate tank and larval density was adjusted to 5 indiv. per liter. Approximately 75% of these larvae survived, and the black wound traces disappeared 4 days after removing. Therefore, we understand that this mass mortality was caused mainly by the direct and indirect effects of initial overstocking. In trial no. 6, the protozoeal larvae that were fed only with an artificial plankton diet had a sudden drop in larval population on the 3rd day was controlled within certain ranges and aeration of rearing, from 100 thousand to 40 thousand was operated strongly in these trials, the fluctua individuals. After that, there was no obvious tion in the observed populations must be due to mass population decline until harvesting. So, we larval size and stocking density.fig. 1. Daily declining patterns can estimate of larval the population apparent on initial a logarithmic stocking scale popula in ten trials.trial nos. 1-3, 4-9, The survival rate of 24-day larval rearing ranged from 0.29 to 0.84 in the ten trials, and were above 0.5 in eight of the trials, the exceptions being trial nos. 6 and 9. As shown in Fig. 1, we drew a straight line between two points (the initial and final populations) in the trials which gave a survival rate of more than 0.5. The observed populations in these trials fluctuated in a small range along a straight line, except for trial no. 3. The larger fluctuation in trial no. 3 is mostly attributed to the diminished accuracy of the volumetrical method of population estimation by the lowest initial stocking population (Table 1). No mass mortality occurrence (critical period) was observed during rearing in these eight trials. Therefore, survival numbers in these trials could be considered to have decreased at a constant daily rate throughout the rearing period. This means that the declining pattern of these popula tions can be expressed by the logarithmic linear equation (dn/dt=-rn; where N is the survival number of the larvae, t is rearing period in days, and r is the daily rate of decline in the survival numbers). There was a sudden drop in population in trial nos. 6 and 9. In trial no. 9, with an initial stock ing number of about 260 thousand individuals, the survival number decreased distinctly from the 13th to the 17th day of rearing (i. e. 4-8th day of the postlarval stage). Several days before the mass mortality occurred, black wound traces were observed in appendages, carapace, and uropod of the larvae. These wound traces were caused by the fish hitting aganinst each other or against the tank wall. Some of the larvae molted incompletely, and swam up and down abnormally with their soft bodies. In order to prove that this occurrence is attributed to overstocking, some of these larvae were removed to a 30l volume tion and survival pattern, based on the assumption that there would have been no mass mortality if we had fed with diatoms at the protozoeal stage as we did in other trials. Using the population data from the 4th day to the final day of rearing, a linear regression through the final population data as a fixed point was statistically determined. The apparent initial population could be estimated by this linear regression equation as about 44 thousand individuals, and the survival rate was 0.72 (Fig. 1).

5 Fig. 2. (a) The relationship between the initial stocking density and the survival rate of the l arvae. The daily declining rate of the populations in rearing runs with less than 32 thousand indiv./ m3 of the initial stocking density has also been noted. (b) Actual values and a calculated qu adratic curve of harvested density in various initial stocking densities. The plotted mark of the trials is the same as in Fig. 1. Including the corrected rearing data of trial no. 6, these ten trials were expected to be operated under good conditions that had been confirmed previously. Therefore, the results of these larval rearing runs were treated as data for elucidating the effect of initial stocking density on harvesting number and yield of the larval production. The initital stocking density and final harvesting density which are noted in this paper were defined by dividing the total survival number of the larvae with a volume of maximal water capacity (5.5m3) of the rearing tank. Results Figure 2a illustrates the relationship between the initial stocking density and the survival rate. This relationship shows a negative correlation, thus increasing the stocking density results in a lower survival rate. The negative correlation between these two variables is expressed by the following equation: S= I(r=-0.828, P<0.01, n=10), whers Sand I are the survival rate of the harvested larvae and the initial stocking density (thousand Fig. 3. (a) The relationship between the initial stocking densities and harvested biomass for each individual of the initial stocked larvae (harvested individual fresh body weight ~survival rate). (b) Yield potential of the larval production with respect to various initial stocking densities, with actual values and the curve of the quadratic equation. Actual values are marked identical to those in Figs. 1 and 2. indiv. /m3), respectively. With an initial stocking density of below 30 thousand indiv. /m3, a survival rate of more than 0.50 is obtained in a 24-day rearing. Final harvesting density (F, thousand indiv. /m3) is estimated by a quadratic equation as follows: F=S EI=( I)I. Actual values and the quadratic curve between the initial stocking density and the final harvesting density are given in Fig. 2b. According to this equation, the largest harvesting density (16.5 thousand indiv. /m3) is obtained at an initial stocking density of approximately 40 thousand indiv. /m3. The weight of harvested biomass (B, mg) by rearing a unit number of initial stocked larvae is calculated by multiplying the final individual fresh body weight with the survival rate. The relationship of the biomass (B) to initial stocking density is shown in Fig. 3a. The correlation is more signif icant than that of the survival rate to the initial stocking density. The regression is described with the following equation: B=W ES= I (r=-0.940, P<0.001, n=10),

6 where W is the individual fresh body weight of the harvested larvae (mg/indiv.). This relationship explains that by increasing the initial stocking density, we obtained less in the biomass increment per one individual of the initial stocked larvae. The maximum yield of the larval production is estimated by the following modified equation: Y=W ES EI=( I)I, where Y is the biomass of the final harvest (yield, mg/m3). According to this equation, the maxi mum yield of larval production is approximately 80g/m3, and the initial stocking density is about 30 thousand indiv./m3. The actual value of the largest yield was 87g/m3 at an initial stocking density of about 25 thousand indiv. /m3 in trial no. 8 (Fig. 3b). The yield of 24-day rearing increases until the initial stocking density of 30 thousand indiv. /m3, and it decreases with a further increase of initial stocking density (the turning point of the quadratic curve). Discussion There has been only one study of P. japonicus that was concerned with the limiting factor of stocking density on pond culture for adult prawns.5) According to this report, stocking density had no direct effect on the survival rate. Generally, prawns reared either in a nursery pond or in a growing pond were expected to survive by % from the stocked number if there were no disease occurrence.1, 12) Wyban et al.4) stated that there was a negative correlation only between the stocking density and growth of P. vannamei adult prawns reared in an earth pond. However, not only the growth but also the survival of prawns reared under intensive culture in closed systems were better at low than at high stocking densities.13, 14) With respect to larval mass production, we perceived that there was a negative correlation between initial stocking density and survival rate (Fig. 2a). Final individual fresh body weight had a negative correlation with final harvesting density (r=-0.617, P<0.05), while it had no correlation to initial stocking density (r=-0.502, P>0.1). These facts imply that larval density effected larval growth mainly from the middle to the latter part of the rearing period. The harvested larvae in trial no. 9, where the larval population once declined drastically in the middle of the rearing period, had a larger fresh body weight than the harvested larvae of trial no. 3 which had a lower initial stocking density than in trial no. 9 (see Table 1). As mentioned earlier, the biomass (B) related more significantly to initial stocking density (P<0.001), tather than to survival rate (P<0.01). This means that survival and growth functions un der these rearing conditions interacted mutually with each other more obviously from the middle to the latter part of the rearing period. Never theless, if the hatched larvae were initially stocked rather at a higher density than that at the ten trails (Table 1), e.g. 400, 500 thousand individuals in a 5.5 m3 tank, the effect of stocking density on growth and survival would certainly work more distinctly in the early period of rearing (i.e. during the developmental stage).15) Larval rearing in this study was conducted for 24 days in a tank with a water capacity of 5.5m3. It is not necessarily concluded that operating under different conditions, e.g. the rearing period, the water capacity of the rearing tank, the water temperature and so on, can obtain a similar result as these rearing trials. However, it is a clear indication that the yield potential of kuruma prawn larval production in closed systems can be illustrated by a quadratic equation modified above (Fig. 3b), if there is no disease occurrence or defective feeding condition. From the rearing conditions of the present study, we found out that thousand indiv. /m3 is the adequate initial stocking density for achieving the largest yield (80g/m3). This value may be referable to the maximum yield of larval production operated with the present rearing techniques (Fig. 3b). Further studies with various rearing factors as mentioned before have to be investigated in order to clarify correspondences in these larval rearing conditions. Hence, a "standard model" for mass larval production techniques can be establish ed. Acknowledgments This research was funded by Nippon Sea Farms Inc. We are very grateful for help given by the staff of Yuya Laboratory, Nippon Sea Farms Inc. References 1) K. Shigueno: Shrimp culture in Japan. Association for International Technical Promotion, Tokyo, Japan, 1975, pp ) M. Hudinaga and J. Kittaka: The large scale production of

7 the young kuruma prawn, Penaeus japonicus Bate. Inf. Bull. Planktol. Japan. Commemoration Number of Dr. Y. Matsue, (1967). 3) K. Shigeno: Intensive shrimp larval culture in Japan. In "The Aquaculture of Shrimp, Prawn, and Crayfish in the World: Basics and Technologies" (ed. by C. Chavez Justo), Midorishobo, Tokyo, 1990, pp (in Japanese). 4) J. A. Wyban, C. S. Lee, V. T. Sato, J. N. Sweeney, and W. K. Richards Jr.: Effect of stocking density on shrimp growth rates in manure-fertilized ponds. Aquaculture, 61, (1987). 5) D. Lanari, R. Ballestrazzi, and E. Tibaldi: Effects of fertili zation and stocking rate on the performance of Penaeus japonicus Bate in pond culture. Aquaculture, 83, (1989). 6) D. I. Williamson: Larval morphology and diversity. In "The Biology of Crustacea" (ed. by L. G. Abele), Vol. 2, Embryology, Morphology, and Genetics, Academic Press, New York, 1982, pp ) E. D. Wood, F. A. J. Armstong, and F. A. Richards: De termination of nitrate in sea water by cadmium-copper reduction to nitrite. J. Mar. Biol. Assoc. U. K., 47, (1967). 8) K. Bendschneider and R. J. Robinson: A new spectrophoto metric determination of nitrite in sea water. J. Mar. Res., 11, (1952). 9) M. Hudinaga: Reproduction, development and rearing of Penaeus japonicus (Bate). Japan. J. Zoo., 10, (1942). 10) K. Mawatari and K. Hirayama: Studies on resistibility of some marine animals at various larval stages to ammonia and nitrite. Bull. Fac. Fish., Nagasaki Univ., (39), 1-6 (1975) (in Japanese). 11) M. P. Hardin, D. L. Hutchins, G. W. Chamberlain, and D. V. Aldrich: Temperature and size effects on the accuracy of estimating postlarval shrimp populations. Aquacul tural Engineering, 4, (1985). 12) AQUACOP, J. Barret, D. Goxe, C. Galinie, and L. Ottogali: Techniques for semi-intensive or intensive rearing of Penaeid prawns/shrimps in nursery and growing ponds. In "The Aquaculture of Shrimp, Prawn, and Crayfish in the World: Basics and Technologies" (ed. by C. Chavez Justo), Mid orishobo, Tokyo, 1990, pp (in Japanese). 13) J. R. M. Forster and T. W. Beard: Experiments to assess the suitability of nine species of prawns for intensive culti vation. Aquaculture, 3, (1974). 14) P. A. Sandifer and T. I. J. Smith: Effects of population density on growth and survival of Macrobrachium rosenbergii reared in recirculating water management systems. In Proc. 6th Annu. Workshop World Maricult. Soc. (ed. J. W. Avault and R. Miller), Louisiana State University Press, B aton Rouge, pp (1976). 15) W. D. Emmerson and B. Andrews: The effect of stocking density on the growth, development, and survival of Penaeus indicus Milne Edwards larvae. Aquaculture, 23, (1981).