Effect of stocking density on the growth and survival of Anomalocardia brasiliana (Gmelin, 1791) (Bivalvia: Veneridae) post-larvae. Effect of stocking density on the growth and survival of Anomalocardia brasiliana (Bivalvia: Veneridae ) post-larvae

: Anomalocardia brasiliana post-larvae were cultured for 28 days to evaluate the effect of stocking density on their growth and survival rate. Three stocking densities were tested 40, 80 and 160 post-larvae cm -2 , in experimental units (2-L) with static cultivation system and total water renewal at each 48 hours. The experimental design was completely randomized with three treatments and three repetitions each. Stocking density of 40 post-larvae cm -2 reached 1 mm length within 24 days, while only 18% of the post-larvae at density of 80 post-larvae cm -2 reached 1 mm length in the same time. The density of 40 post-larvae cm -2 had the highest specific growth rate, 4.98±0.08% day -1 . The survival rates of post-larvae at lower densities were significantly higher (53.24±4.60% and 52.95±3.32%, on 40 and 80 post-larvae cm -2 , respectively) compared to higher stocking density (31.54±0.70%). In the cultivation of A. brasiliana post-larvae, stocking density must be managed during growth. The density of 160 post-larvae cm -2 can only be used until the post-larvae reach 600μm in length, then they should be at 40 post-larvae cm -2 density to keep the maximum daily growth rate.


INTRODUCTION
Mollusk production is an important activity within world aquaculture. Although, not a recent activity, mollusk production still has obstacles such as the lack of proper methodologies for domesticating native species and economic viability of production (FAO, 2018). In Brazil, the main mollusk specie produced is Crassostrea gigas, a temperate oyster that, when grows at high temperatures, growth and survival rates may be compromised (POLI, 2004). To make oyster production feasible in the tropical regions of the Brazil, native species such as Crassostrea gasar (CHRISTO & ABSHER, 2006;LEGAT et al., 2017) and Crassostrea rhizophorae (GALVÃO et al., 2009;LOPES et al., 2013) are being studied. Moreover, the Brazil stands out in the scenario of mollusk production for being the world's most important producer of the brown mussel Perna perna (~20,000 tones year -1 ; FAO, 2018).
In addition to the production of oysters and mussels, from the 1990s clams aquaculture developed rapidly throughout the world and contributed to the growth of mollusk production (FANG & LIN, 2016). However, variations in environmental parameters and larvae availability have already caused oscillations in clams world production (e.g. Ruditapes spp. and Tapes philippinarum; HUO et al., 2018). In Brazil, Oliveira et al. oscillations in clams availability were related to atypical environmental conditions and overfishing, since no management plan has yet been decided. This irregularity has been causing social and economic impacts (SILVA-CAVALCANTI & COSTA, 2009;CORTE et al. 2017).
Anomalocardia brasiliana (Gmelin, 1791) is the most relevant clam for Brazilian fisheries, mainly in Northeastern Brazil (SILVA-CAVALCANTI & COSTA, 2009;OLIVEIRA et al., 2011;LAVANDER et al., 2011;OLIVEIRA et al., 2014). It belongs to the Veneridae family and found in place of lives at water depths ranging from 0.5 to 1.5 m into shallow sandy grounds near mangroves. It is sensitive to environmental variations; high mortality levels are noted during the rainy season which causes large fluctuations in the size and distribution of populations (MONTI et al., 1991;MOUËZA et al., 1999;FUNO et al., 2019).
Ideal stocking density for A. brasiliana has been extensively studied. However, most studies have evaluated the growth period in the D-larvae phase to metamorphosis pediveliger (LIU et al., 2010;OLIVEIRA et al., 2014;LIMA et al., 2018), as they are critical stages; larvae are fragile and susceptible to diseases during handling. After the metamorphosis pediveliger, postlarvae become more resistant to handling and can be grown at higher densities, for a longer period (days) until they reach seed size (1 mm), when they can be transferred to the field (NICOLAS & ROBERT, 2001;EPELBAUM et al., 2011;LIU et al., 2010).
The evaluation of the stocking density during the post-larval stage aims to find the ideal environmental conditions for the post-larvae to reach seed size in the shortest possible time. In this sense, the present study aimed to evaluate the effect of stocking density on the growth and survival rate of Anomalocardia brasiliana post-larvae in a closed cultivation system.

Breeding collection and acclimatization
A total of 500 Anomalocardia brasiliana brooders (24.50±2.00 mm length) were collected at the beach of Mangue Seco, state of Pernambuco, Brazil (07º50'1.19"S, 034º50'39.1"W). Brooders were transported to Sustainable Mariculture Laboratory (LAMARSU), linked to the Federal Rural University of Pernambuco. There, the animals already in the final stage of maturation were acclimatized in 500-L tank, during 24 h and a density of 1 ind L -1 , where water quality was under control: temperature (25 °C), 30-salinity and dissolved oxygen medium (6 mg L -1 ), and fed with Chaetoceros calcitrans (300,000 cells mL -1 ).

Release of gametes and larviculture of A. brasiliana
After acclimatization, the brooders were induced to release gametes, by raising the temperature to 28 °C and after that, adding C. calcitrans (300,000 cells mL -1 ), in a 400-L tanks in stocking density of 4 larvae L -1 . After the release of gametes, the eggs were collected in 50 µm-meshes, counted and placed in 30-L incubators with filtered seawater at 1.5 µm and sterilized with sodium hypochlorite at 30-salinity and total renewal of water every 48 hours.
Eggs were kept in the conical incubators for 15 days until reaching the post-larval stage; in this step, a density of 10 larvae mL -1 was adopted. The larvae were fed with a mixture of microalgae, C. calcitrans and Pavlova lutheri, with cells ratio of 1:1 using different algal concentration. From the first to the seventh day 30,000 cells mL -1 ; from the eighth to the twelfth day 40,000 cells mL -1 and; from thirteenth day until the start of the experiment 50,000 cells mL -1 was used (OLIVEIRA et al., 2014). The feeding was adjusted by observing the algal consumption, by daily counts of residual algal using a Neubauer chamber. In addition, water quality also was under control: temperature (25±1 °C), 30-salinity and dissolved oxygen (over 5 mg L -1 ).

Experimental design and management of post-larvae
At the beginning of the experiment postlarvae with 15-day-old were measured (n = 30) and had an average size of 307.89±50.92 µm in length (measured between the anterior and posterior ends of the shell). Post-larvae were transferred to experimental units (2-L plastic containers), which had a center 10 cm PVC cylinder (area 78.5 cm²) was placed in suspension, with a mesh of 250 µm in the bottom, and an airlift for water circulation ( Figure 1). The post-larvae were cultured for 28 days, in three different stocking densities: 40, 80 and 160 postlarvae cm -2 , with three replicates each, in a closed water circulation system with full exchange at each 48 hours. This system proved to be efficient for the three densities tested and it was possible to maintain water quality at optimum levels for this species: temperature (25 °C), 30-salinity and dissolved oxygen (over 5 mg L -1 ). During the experiment, the feeding was composed by C. calcitrans and P. lutheri and offered daily, in the same cell ratio (1:1), using algal concentration of 50,000 cells mL -1 , over 28 days.

Post-larval development
For the evaluation of post-larval growth, 1mL samples were taken from experimental units every 7 days. Images were taken by a camera attached to a light microscope (Coleman -N107 LED), using a Sedgewick-Rafter chamber. Each week, random photos of 30 post-larvae of the experimental units were taken. For evaluating the growth of post-larvae, length measurements (maximum antero-posterior dimension) (OLIVEIRA & OLIVEIRA, 1974) were taken with the software ImageTool version 2.0 (Texas University, Health Science Centre, San Antonio, USA).
The growth rate of post-larvae was estimated using the following formula (LIU et al. 2006): SGR = 100 (lnL2 -lnL1) / T, where SGR stands for specific growth rate (% day −1 ); L1, L2, stand for shell length at the beginning and at the end of each experiment day, respectively, and "T" duration of experiment (days).
At the end of experiment, the surface coverage rate (SCR) was estimated from the covered area (AO): AO= [(L/2) × (H/2) × π] × N where AO is the area occupied by shells, L is the average length of the shells, H is the mean height and N is the total number of post-larvae per experimental unit. The result was correlated to the background area (78.5 cm²) to find the SCR (%), methodology by LIU et al. (2010).
For the evaluation of post-larval survival at the end of the cultivation, the entire volume of each experimental unit was filtered, concentrating the postlarvae in 50 mL containers, from which three samples of 1 mL were taken; the individuals were counted using a Sedgewick-Rafter chamber.

Statistical analysis
The survival data, length and growth, was previously checked for normality using the Kolmogorov-Smirnov test and the Cochran C homogeneity of variance test. Analysis of Variance (ANOVA one-way) was used to determine the effect of density on the growth and on the survival of post-larvae among the sampling intervals (7 days). Tukey's test was performed to detect how the treatment interfered in the average results obtained, between treatments differ significantly, at a significance level of 5% (p<0.05). Data are presented as mean ± standard error.

RESULTS
Average lengths of A. brasiliana postlarvae stocked at different densities at 7-day intervals are shown in figure 2. Differences (P>0.05) in lengths of post-larvae at 40 post-larvae cm -2 were identified on the 14 th day of cultivation (length 814.54±56.71µm). The post-larvae at the highest stocking density (160 post-larvae cm -2 ) had lower average lengths (P<0.05) at the end of the 28 th day of cultivation, with 791.21 µm of average lengths; while the other lengths, different from each other (P<0.05), were 1,249.77 µm (40 post-larvae cm -2 ) and 972.71 µm (80 postlarvae cm -2 ). On the 24 th day, all post-larvae cultivated at 40 post-larvae cm -2 density reached 1,000 µm in length; 18% of the post-larvae grown at 80 postlarvae cm -2 reached the same length after 28 days of cultivation, and post-larvae grown at 160 post-larvae cm -2 did not reach this length at all (Table 1). The same was observed when assessing the height, the treatment differed significantly (P<0.05) from each other and the one with the highest density (160 post-larvae cm -2 ) presented the lowest height. Average larval survival final at lower densities (40 and 80 post-larvae cm -2 ) was 53.24±4.60% and 52.95±3.32%, respectively, differing significantly from the rate at the highest stocking density (31.54±0.70%).
The highest SCR was reported in 80 postlarvae cm -2 (28.83%). Because of the high mortality of the treatment with 160 post-larvae cm -2 , the SCR value of this treatment did not differ (P>0.05) from the treatment with lower density, being 25.06% and 23.27%, for 40 and 160 post-larvae cm -2 , respectively. Table 2 shows the post-larvae SGR at the three different stocking densities. The fastest growth occurred in the first seven days of the experiment. The lowest density (40 post-larvae cm -2 ) showed the highest growth rate until the fourteenth day, differing from the two other densities evaluated (P<0.05). After 21 days of cultivation, growth rate dropped sharply at all three densities (Table 2).

DISCUSSION
Food is a growth limiting factor of bivalve mollusc cultured in high densities (LOOSANOFF & DAVIS 1963;SPRUNG, 1984). To study the effect of stocking density on a target species, one should ensure that food does not exceed a maximum and minimum desirable concentration (LIU et al., 2010). In this study, the residual algal was checked daily, and adjusted as necessary, to maintain the same concentration in each dietary treatment and ensure that food was not lacking, especially at the highest density. Thus, it is assumed that food was not a limiting factor.
In the present study, it was observed that the stocking density impairs the development of post-larvae and the greatest growth was obtained at a density of 40 post-larvae cm -2 , with 1,249.77 µm and 1,156.31 µm in length and height, respectively. Once, high stocking densities in the cultivation of bivalve post-larvae can cause reduced growth. This can be attributed not only to food shortage, but also to low confinement space, which will cause deterioration in the water quality (RAGHAVAN & GOPINATHAN, 2008; VELASCO & BARROS, 2008). In addition, high stocking density causes discontinuous consumption of food resulting from frequent collisions among the post-larvae, since the post-larvae close their valves and stop feeding due to the mechanical shock (LOOSANOFF & DAVIS, 1963;LIMA et al., 2018), impairing survival and growth of the post-larva. In this study, probably, the continuous water flow, together with increased stocking density, contributed to the increase of the post-larvae collisions.
In post-larvae culture of A. brasiliana, LIMA et al. (2018) when testing the density of 60 and 120 post-larvae cm -2 , also observed that survival and growth of organisms is influenced by the density of culture. These authors reported survival and growth of 93.81% and 2,177.34 µm, for the density of 60 post-larvae cm -2 , which resulted in a 48% increase in length. OLIVA et al. (2014) studied the effect of density on growth and survival, during the initial larval phases and after establishment of the species M. edulis in the laboratory. The authors observed that the density affected the growth of the species, since post-larvae cultured at 5 pediveliger cm -2 density were 28% larger than the post-larvae cultured at 10 pediveliger cm -2 . The best results for survival were also obtained at lower densities, reaching up to 68%. These results agree with the present study, where the density affected the growth and the survival during the post-larval phase of A. brasiliana.
The static system with total water exchanges every 48 hours is commonly used in larval and post-larval bivalve cultivation (ROBERT & GERARD, 1999). RAGG et al. (2010) recommend the continuous water flow to larviculture using smaller volumes and high densities of larvae or postlarvae. The continuous flow cultivation system is more efficient than the static system, with or without changing the water, presenting in general higher survival results (TURINI et al., 2014); however, even in this system, the high density affected the yield of Anomalocardia brasiliana post-larvae in this study.
Studies showed that the background area can limit density for larvae with benthic habits. In post-larvae foot there is a reduction in growth when subjects occupy 100% or more of the bottom coverage area of the experimental unit (LIU et     Oliveira et al. al., 2010). HEASMAN et al. (2002) noted that an occupation of 70% of the bottom area is a limiting factor for the development of juvenile scallops (Pecten fumatus) as density is a limiting factor of their growth rate. For Clinocardium nuttallii however, a full use of the bottom area (100%) is possible, if individuals are not longer than 1 mm in length; in such case, a density of 160 post-larvae cm -2 would be recommended (LIU et al., 2010). In the present study, the bottom coverage area was not a growth limiting factor to A. brasiliana, it was observed that, due to the high mortality, the treatment with 160 post-larvae cm -2 presented similar SCR values of the treatment with low density and the higher SCR was obtained in 80 post-larvae cm -2 , at the end of cultivation.
The specific growth rate of Meretrix meretrix larvae was lower than 2% when they were in the period of metamorphosis and settlement, regardless of the stocking density (LIU et al., 2006). In the case of A. brasiliana post-larvae; although, it was at a different stage, there was a drop in SGR (<2%) on the 21 st day. After 24 days of cultivation, post-larvae at the lowest density (40 post-larvae cm -2 ) reached 1 mm in length. Such growth was not achieved in other densities; post-larvae growth rate at higher densities was lower, affecting the final size of individuals at the end of the cultivation. If a 600 µm length is desirable, a density of 160 post-larvae cm -2 can be used. If a 1 mm length is the target, the suitable density is of 40 post-larvae cm -2 . In both cases, the maximum growth rate would be maintained.

CONCLUSION
Stocking density directly affects growth and survival rates of settled A. brasiliana post-larvae. In a closed culture system, the density of 160 postlarvae cm -2 can be used until the post-larvae reach 600μm in length, then they should be in density of 40 post-larvae cm -2 to maintain the specific growth rate at around 5% per day.

ACKNOWLEDGMENTS
This study had the support of Brazilian National Counsil for Scientific and Technological Development (CNPq) for the aid granted to the Professor Alfredo Olivera Gálvez (PQ 311058/2015-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil -Finance code 001, and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE).

ETHICS AND BIOSSECURITY COMMITTEE APROVAL
The study did not require the approval of an institutional ethics committee, since the work was developed with oysters (bivalve mollusks), not falling under Art. 2 of Law 11.974 of October 8, 2008: "The provisions of this Law apply the animals of the species classified as phylum Chordata, subphylum Vertebrata, observing the environmental laws".