Aquacult Int DOI 10.1007/s10499-017-0140-3
Effects of temperature, salinity, diet and stocking density on development of the veined Rapa whelk, Rapana venosa (Valenciennes, 1846) larvae Tao Zhang 1,2 & Hao Song 1,3 & Yu-Cen Bai 4 & Jing-Chun Sun 1,2 & Xiao-Fang Zhang 5 & Shao-Jun Ban 6 & Zheng-Lin Yu 1,3 & Mei-Jie Yang 1,3 & Hai-Yan Wang 1,2
Received: 21 October 2016 / Accepted: 14 March 2017 # Springer International Publishing Switzerland 2017
Abstract The veined rapa whelk Rapana venosa (Valenciennes, 1846) is an important and valuable fishery resource but has not been cultured on a large scale. We studied the effects of environmental factors, temperature, salinity, diet, and stocking density, on growth and survival of larvae to determine optimal artificial culture conditions. The optimal temperature was 25– 31 °C at 30 ppt at densities of about 200 veligers per liter, under which the mean shell length (737–1006 μm) and survival rate (36–45%) were higher than those held at other temperatures. When cultured under a salinity of 25 ppt at 25 °C with densities of about 200 veligers per liter, larvae had the highest mean shell length (878.45–917.88 μm) and survival rate (29.75%). Larvae were fed a mixed diet, Pseudoisochrysis paradoxa + Tetraselmis chui + Chlorella vulgaris. Optimal stocking density was 300 veligers per liter for the first 5 days and was 100 veligers per liter afterward at 25 °C and 30 ppt. Keywords Rapana venosa . Gastropod . Larva . Development
* Hai-Yan Wang
[email protected]
1
CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, China
2
Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
3
University of Chinese Academy of Sciences, Beijing 100049, China
4
China Rural Technology Development Center, Beijing 100045, China
5
Zhangzidao Island Fishery Group Limited Company, Dalian 116001, China
6
Laixi Environmental Protection Bureau, Qingdao 266600, China
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Abbreviations SL Shell length SR Survival rate GR Growth rate
Introduction Rapana venosa belongs to the Class Gastropoda, Family Muricidae, Genus Rapana (Kool 1993). It is native to temperate Asian waters, the Sea of Japan, Yellow Sea, Bohai Sea, and the East China Sea to Taiwan in the south (Mann and Harding 2003). R. venosa is an important and valuable fishery resource in its native habitat; however, R. venosa was overexploited and its habitat was destroyed in some locations, which resulted in severe population declines (Yang et al. 2007; Wei et al. 1999). The planktonic larva period is one of the most vulnerable phases of the rapa whelk life cycle (Harding 2006), and as a result, this phase is critical for increases in the population. Thus, it is necessary to understand the larval development process, establish technical procedures for R. venosa culture, and avoid overexploitation. Among all of the environmental factors, temperature, salinity, diet, and stocking density are considered to be the most important factors influencing developmental times and survival rate of larvae in culture (Liu et al. 2009, 2010; Tang et al. 2012; Wang et al. 2012) and are easier to be measured and controlled than others in artificial larval production systems. Temperature is an important factor regulating the rate of all biological processes such as survival, length of pelagic life, and metamorphosis (Scheltema 1967). Although temperature is an important modifier of larval growth, salinity imposes the greatest additional load on metabolic requirements. During the time when ambient salinity varies, cells and tissue of larvae will consume large amounts of energy to adjust osmotic pressure (Bao and You 2004). The change in osmotic pressure will also decrease metabolic speeds and influence metabolic efficiency (Bao and You 2004). Food conditions can affect development, condition, and survival of larvae (Basch 1996). Furthermore, the cost of feed can represent up to 40% of the overall production costs in aquaculture (Woodcock and Benkendorff 2008). The purpose of commercial larviculture is to increase production per unit in a limited water volume at minimal cost, which is related to larval density. Thus, these four factors were selected for the present study. Many studies have been carried out regarding the effects of the environment on growth and survival of molluscs and larval-rearing techniques (Ibarra et al. 1997; Liu et al. 2006; Liu et al. 2010; Wang et al. 2012). In addition, the culture of R. venosa benthic juveniles and adults has been studied by Wang et al. (1997) and Yang et al. (2007); nevertheless, there is a paucity of information on the culture of larvae. The studies on R. venosa larvae mainly focused on basic biological questions, such as the reproduction cycle (Mann et al. 2006; Saglam and Duzgunes 2007; Westcott et al. 2001), egg capsules, and larval development process (Lee 1959; Wei et al. 1999). Moreover, some studies on the effect of salinity on embryo and larval development (Mann and Harding 2003; Wang et al. 2003) of R. venosa have been undertaken. No previous studies related to feeding behavior, nutritional needs, stocking temperature and density of the R. venosa post-hatching stage are available. Correspondingly, larvae have never been artificially cultured on a large scale, and their growth and survival requirements under captive conditions are not fully understood. In this study, experiments were designed to develop commercial hatchery rearing techniques, reveal optimal conditions for the highest
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growth and survival rates in artificial rearing system of R. venosa, and increase production per unit in a limited water volume with minimal cost.
Materials and methods Adult conditioning R. venosa broodstock were collected from Haizhou Bay (Rizhao, China) and gradually conditioned in seawater from 13 to 25 °C for several weeks prior to the laying of egg capsules. Egg capsules were collected and then cultured in a tank at a temperature of 25 °C and salinity of 30 ppt. After hatching, larvae were collected within 24 h and used for experiments.
Structure of the experiment Seawater was filtered through gravel and sand and kept under continuous aeration to supply oxygen. Thermostatted heaters were utilized to control the temperature of seawater within ±0.5 °C. Low salinities (5, 10, 15, 20, and 25 ppt) were obtained by diluting seawater with fresh water which was aerated for at least 1 day and filtered through the screen with 39-μm meshes before utilization. High salinities (35, 40, and 45 ppt) were obtained by mixing seawater with sea salt. To eliminate waste, seawater in all cultures was changed each day, but experimental conditions were kept unchanged and consistent. Except for the diet experiments, veligers were fed with a mixed diet of Pseudoisochrysis paradoxa, Chlorella vulgaris, and Tetraselmis chui every day (2 × 104–4 × 104 cell/ml). In order to keep salinities constant in the salinity experiment, algal food was added only before the adjustment of salinities. Based on preliminary tests, it was found that 25 °C was the optimal temperature for larval growth. Moreover, salinity of seawater utilized was 30 ppt, which was consistent with the broodstocks’ natural oceanic habit. Thus, egg capsules were hatched under conditions of a temperature of 25 °C and salinity of 30 ppt. Larvae were cultured under condition of temperature of 25 °C and salinity of 30 ppt, except for the temperature in temperature experiments and salinity in salinity experiments; however, temperature and salinity should be gradually modified in temperature (5 °C/day) and salinity (3 ppt/day) experiment, respectively. Larvae which were from the same broodstock were collected by a screen with 235-μm meshes within 24 h after hatching and then were resuspended in experimental buckets.
Effect of temperature on larval growth and survival rate The lowest temperature egg capsules were laid at 16 °C. Thus, the designed temperatures ranged from 16 to 34 °C at 3 °C intervals with the salinity at 30 ppt at densities of about 200 veligers per liter. Three replicates were established in each treatment. Larvae were initially cultured in 10-l polypropylene buckets with seawater at a temperature of 25 °C and salinity 30 ppt. Then, temperature was adjusted at a rate of 3 °C/day. Average initial shell length of larvae was 365 ± 3 μm.
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Effect of salinity of larval growth and survival rate Broodstock of R. venosa laid egg capsules in the area of the estuary where the salinity is low. Larvae grow up in shallow seas where the range of salinity is wide. Thus, growth and survival rate were studied in three replicates at nine salinities ranging from 5 to 45 ppt at 5 ppt intervals at temperature 25 °C with densities of about 200 veligers per liter. Larvae were initially cultured in 10-l polypropylene buckets with seawater at a temperature of 25 °C and salinity of 30 ppt; then, salinity was adjusted at a rate of 5 ppt/day. Average initial shell length of larvae was 351 ± 2 μm.
Effect of different algal food species on larval growth and survival rate The diet experiment was carried out to determine whether different algae had an effect on growth and survival rate of larvae. Average initial shell length of larvae was 345 ± 2 μm. The larvae were reared in 10-l polypropylene buckets with seawater at a temperature of 22 °C and at salinity 30 ppt at densities of about 200 veligers per liter. The diets were (a) Pseudoisochrysis paradoxa, (b) Chlorella vulgaris, (c) Tetraselmis chui, (d) Pseudoisochrysis paradoxa + Chlorella vulgaris(1:1), (e) Pseudoisochrysis paradoxa + Tetraselmis chui (1:1), and (f) Pseudoisochrysis paradoxa + Chlorella vulgaris + Tetraselmis chui (1:1:1). Each treatment included three replicates (2 × 104–4 × 104 cell/ml.
Effect of larval density on larval growth and survival Ten stocking densities were designed: 50, 100, 200, 300, 500, 800, 1000, 1500, 2000, and 3000 individuals/l. Larvae were reared in 10-l polypropylene buckets with seawater at a temperature of 25 °C and salinity 30 ppt. Each treatment included three replicates. Average initial shell length of larvae was 365 ± 3 μm. Mixed diet (Pseudoisochrysis paradoxa, Chlorella vulgaris, and Tetraselmis chui, 2 × 104–4 × 104 cell/ml) proportionately increased with stocking density to ensure larvae in each treatment received an equal diet.
Methods of taking samples and statistical analysis Samples were taken to record shell length (the maximum dimension), growth rate, and survival rate of larvae at 0, 5, 10, and 15 days in temperature experiments and salinity experiments, at 0, 5, 10, 15, 24, and 27 days in diet experiments and at 0, 5, and 10 days in density experiments. Larvae in each culture were filtered through a screen, respectively, and transferred to petri dishes. After draining the seawater in petri dishes, the larvae stopped swimming and the shell length was measured under a microscope. Thirty larvae were measured in each culture. After measurement, the larvae were returned to their original culture. Mean shell lengths (SL) of larvae were calculated between three replicate cultures. Daily growth rates (GRs) of larvae were calculated using the formula: GR ¼ ðSLt −SLt‐5d Þ=5d where t is the time post-hatch and SL is the mean shell length of larvae. To investigate the effects of temperature, salinity, diet, and density on the survival of larvae, respectively, a cup of seawater which was churned to make the larvae evenly distributed was
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taken to measure the volume of seawater and count the number of larvae in it, to calculate larval destiny. Each measurement was carried out three times in each treatment. Mean larval density of three replications (D) was obtained and the larval survival rate (SR) was estimated by the formula: SR ¼ ½ðDt V Þ=ðDt0 V Þ 100 where t is the time post hatch, t0 is 0 day, and V is the volume of seawater in polypropylene bucket. One-way analysis of variance (ANOVA) was used to test the respective effects of temperature, salinity, diet, and density, and differences between means were compared using LSD test with a 95% confidence interval.
Results Effect of temperature on larval growth and survival rate SL (μm) with standard errors of R. venosa larvae at different temperatures is shown in Fig. 1. During the sampling period, the largest SL was observed at 31 °C, while the smallest was observed at the lowest temperature, 16 °C (P < 0.01). Meanwhile, the observed values of SL were higher at 31 °C than that at higher and lower temperatures. Analysis indicated that there were significant differences of SL among different temperatures from day 5 (P < 0.05). In the entire experimental period, SL at 31 °C was significantly longer than the others (P < 0.01). SL values at 28 and 34 °C, compared with the SL at 31 °C, had no significant difference (P > 0.05), but both of them had significant difference with others (P < 0.01). From days 5 to 10, SL at 16 and 19 °C had no significant difference (P > 0.05), but both of them were significantly lower than other treatments (P < 0.05). On day 15, there was no significant difference among 16, 19, and 22 °C (P < 0.01), but all of them were significantly lower than others (P > 0.05). Figure 2 shows the SR (%) of different temperature groups on each sampling day. The optimal SR occurred at 28 °C from day 5 to day 10 and at 31 °C on day 15, and the value of SR decreased when temperature became either higher or slower. An exception was that the SR value at 16 was higher than that at 25 on day 10 and SR value was higher than that at 22 on day 1150
Mean s h ell len g t h (µm)
Fig. 1 Mean shell length (μm) of larval R. venosa cultured at different temperatures. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.05)
16 ć 19 ć 22 ć 25 ć 28 ć 31 ć 34 ć
950
750 550
350 150 0d
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10d
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16 ć 19 ć 22 ć 25 ć 28 ć 31 ć 34 ć
100
Su rv iv al rat e (%)
Fig. 2 Survival rates of R. venosa larvae cultured at different temperatures. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.05)
80 60
40 20 0
0d
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15. Analysis indicated that the difference of SR at different temperatures was not statistically significant (P > 0.05) on day 5. From day 10, a statistical difference began to appear at different temperatures (P < 0.05). However, on day 10, between adjacent temperature treatments (16–19, 19–22, 22–25, 25–28, 28–31, 31–34 °C), no significant differences were observed except between 16 and 19 °C.
The effect of salinity of larval growth and survival rate Figure 3 shows the SL (μm) of R. venosa larvae at different salinities on day 20. The longest and shortest SL occurred at salinity of 20 and 35 ppt, respectively. In addition, the value of SL at 20 ppt had no statistical difference with that at 25 ppt (P > 0.05). The value of SL at 30 ppt had no significant difference with that at 10 ppt (P > 0.05). For all other salinities, the values of SL at different salinities were significantly different with each other (P < 0.05). GRs (μm/day) with standard errors of R. venosa larvae at different salinities on each sampling day are shown in Fig. 4. From day 0 to day 10, the GR of all salinities increased, except salinity 5, 40, and 45 ppt on day 10. In contrast, from day 10 to day 15, the GR of all salinities decreased except the GR at salinity 20 ppt which increased over the experimental period. In addition, the GRs of salinity 45 ppt on day 10 and 40 ppt on day 15 were negative. Fig. 3 Mean shell length (μm) of R. venosa larvae at different salinities on day 20. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
Aquacult Int Fig. 4 Growth rates of R. venosa larvae cultured at different salinities. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
Figure 5 shows the SR (%/day) of R. venosa larvae at different salinities on each sampling day. From day 5, the significant difference of SR began to appear at the different salinities (P < 0.05). On day 5, the value of SR at salinities 20 and 30 ppt had significant differences with that of salinities 15, 25, and 40 ppt (P < 0.05), and there was no significant difference among the other treatments. On day 10, the SR of salinities 5, 40, and 45 ppt decreased rapidly. The lowest SR occurred at a salinity of 45 ppt which had a significant difference with the other salinities (P < 0.05). In addition, the SR from salinity 10 to 35 ppt decreased slowly, and there was no statistical difference among them. On day 15, SR of salinity 45 ppt dropped to 0. The slowest decline and highest values of SR were observed at salinities 10, 15, and 25 ppt, and there were no significant differences among them (P > 0.05). On day 20, the SR in salinities 5, 35, and 40 ppt dropped to 0. The best SR occurred at salinity 25 ppt which had no significant difference with 10 ppt.
Fig. 5 Survival rates of R. venosa larvae cultured at different salinities. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
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Effect of different food algal species on larval growth and survival rate During the diet experiment, the highest SL was observed at group F except on day 10, and the SL at group B was always lowest (Fig. 6). It was noted that from the 15th day, the diets did not contain Tetraselmis chui which resulted in the poorest SL (P < 0.01) among all the diets tested. On the other hand, from day 20, the groups fed with both of Pseudoisochrysis paradoxa and Tetraselmis chui, i.e., groups E and F, exhibited a relatively higher SL. At the end of the dietary experiment, larvae fed with Pseudoisochrysis paradoxa + Tetraselmis chui + Chlorella vulgaris (1:1:1) had the highest growth rate, followed by those fed with Pseudoisochrysis paradoxa + Chlorella vulgaris (1:1), and were significantly higher than those fed with others (P < 0.01). Figure 7 shows the SR of R. venosa larvae, fed with various algal species, on each sampling day of the diet experiment. Over the entire experimental period, SR of larvae fed with Pseudoisochrysis paradoxa + Chlorella vulgaris (1:1) was the highest, but had no significant difference with larvae fed with Pseudoisochrysis paradoxa + Tetraselmis chui + Chlorella vulgaris (1:1:1) (P > 0.05), except on day 15 when larvae fed Pseudoisochrysis paradoxa + Chlorella vulgaris (1:1) were significantly different to larvae fed with Pseudoisochrysis paradoxa + Tetraselmis chui + Chlorella vulgaris (1:1:1) (P < 0.05). From day 10, the shortest SR was shown by larvae fed with Pseudoisochrysis paradoxa + Tetraselmis chui (1:1). It was noted that starting from the 15th day, the SR was lowest in those diets containing no Chlorella vulgaris, i.e., C and E, whereas the SR was higher in the diets comprising both of Pseudoisochrysis paradoxa and Chlorella vulgaris, i.e., groups D and F.
Effect of larval density on larval growth and survival The growth and survival rates were always kept at a high level when R. venosa larvae cultured at low density, but they would drop sharply when the density exceeds a threshold. As shown in Fig. 8, the value of SL of the larvae which were cultured at densities of 1000, 1500, 2000, and 3000 individuals/l on day 5 was lower than the size at hatching
Fig. 6 Mean shell length of R. venosa larvae cultured under different diets. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
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Fig. 7 Survival rates of R. venosa larvae cultured under different diets. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
time. This maybe because the number of dead larvae with longer SL was larger than that with shorter SL. Statistical difference in SL at different stocking densities appeared from day 5, and greater differences occurred with increased culture time. On day 10, larvae reared at the highest density, 800 individuals/l, had the smallest mean size (418 ± 0.6 μm), whereas larvae at the lowest density, 50 individuals/l, had the largest mean size (537 ± 0.6 μm). Figure 9 shows that the larvae which were cultured at densities of 1000, 1500, 2000, and 3000 individuals/l died within 5 days. The threshold survival value was at approximately 800 individuals/l. SR was negatively correlated with density. SR was dependent on stocking density when there were greater differences between densities, whereas independent of stocking density when the densities were less different.
Fig. 8 Mean shell length of R. venosa larvae cultured at different stocking densities. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
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Fig. 9 Survival rates of R. venosa larvae hatched at stocking densities. Bars show the standard errors. Means with the same symbol are not significantly different (P > 0.01)
Discussion The effect of temperature on larval growth and survival rate Growth rate of larvae was found to be dependent on temperature. Previous studies of Pinctada margaritifera showed that growth rate was positively correlated with temperature up to a point at which further temperature increases resulted in a declining growth rate (Doroudi et al. 1999). Similar result was found in our study. SL increased with increasing temperature from 16 to 31 °C. This increase of larval growth was probably caused by the more frequent activities of velum cilia which were used for swimming and ingestion when larvae were under a higher but suitable temperature (Davis and Calabrese 1964). Moreover, the energy consumed on metabolic regulation decreased as the temperature increased; thus, more energy could be used for development (Rico-Villa et al. 2009). In contrast, growth rate significantly decreased with a further increase in temperature to 34 °C. That may be because that temperature was so high that water quality had deteriorated (Liu et al. 2009) and algae was destroyed (Calabres 1969; Ukeles 1961). The deteriorated water quality and destoryed algae could lead to reduced growth rates of larvae. In the present study, survival rate of larvae increased with increasing temperature and then decreased with a further increasing temperature except at 16 °C. It is noticed that survival rate at a temperature of 16 °C was significantly higher than that at 19 and 22 °C (P < 0.05). According to historical studies on structural changes of digestive system of larval Octopus maya and Dicologoglossa cuneata, larval development was divided into three stages: the endogenous feeding (endotrophic) stage, mixed feeding stage, and exogenous feeding (exotrophic) stage (Herrera et al. 2010). Larvae of R. venosa hatched with yolk which could provide energy for survival of larvae. Therefore, at the beginning of post-hatching phase, the development stage of digestive system of R. venosa larva may be the mixed feeding stage and acquired energy from both of the yolk and algae. Moreover, Ye et al. (2008) indicated that the oxygen consumption rate of mature R. venosa increased sharply between 18 and 24 °C. Thus, for mature R. venosa, energy expenditure on respiration increased sharply between 18 and 24 °C. Unfortunately, there is no similar data for larvae.
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Results of our study showed that growth rate of larval reared at 22 °C was significantly faster than that at 16 °C. This means that larval energy consumed on growth was higher at 22 °C than 16 °C. Thus, it is postulated that the energy consumption of larvae cultured at 16 °C was lower than larvae cultured at other temperatures, which means that larvae cultured at 16 °C need less energy coming from food to maintain a positive energy balance. Activity of the enzyme system and filtration rate may not be high enough at the lower temperature; as a result, larvae could not acquire greater energy input through an increased rate of algal digestion. Effects of low energy input on larvae may be less at 16 °C than that at 19 and 22 °C. Thus, survival rate at a temperature of 16 °C was higher than that at 19 and 22 °C. But, the real reason for this difference should be studied further.
Effect of salinity of larval growth and survival rate Growth and survival rate are positively correlated with salinity up to a peak point, after which a further increase in salinity will result in decrease in growth and survival rate. Similar observations with bivalve species have been recorded in the literature: Pinctada martensii (Wang et al. 2012) and Argopecten irradians (He and Zhang 1990). That may be because that the increase or reduction of salinity will increase metabolism which can be related to the energetic cost of osmotic adjustment. In addition, in this study, growth rate decreased and mortalities occurred at all salinities thorough experiment, but were especially pronounced at higher salinities. This suggests that R. venosa faces the greatest osmoregulatory challenge under higher salinities. But, in some species of bivalve larvae, growth rate at low salinity decreased quickly (Lough and Gonor 1971; Tettelbach and Rhodes 1981). According to our study, optimal salinities for growth occurred at 20 to 25 ppt, and for survival, it is at 10 and 25 ppt. Moreover, the lower salinity limit was not reached in our research, although the lowest salinity was 5 ppt and the upper salinity limit was 35 ppt. Results of our study are consistent with results of Mann and Harding (2003), although the methods of these two studies were different. Mann and Harding (2003) designed a series of 48-h salinity tolerance experiments at salinities from 7 to 32 ppt, which were completed using larvae in ages ranging from immediately post-hatch through the onset of settlement (Mann and Harding 2003); in our study, the larvae used were at age of zero day and reared in a series salinities from 5 to 45 ppt until the end of research.
The effect of different food algal species on larval growth and survival rate The results presented in this study indicated that growth and survival rate were affected by the types of food available to the organisms. SR of larvae which were not fed with Chlorella vulgaris was lower than that of larvae which were fed. Similarly, larvae fed with Tetraselmis chui had a longer SL than larvae fed with no Tetraselmis chui. That may mean that Chlorella vulgaris and Tetraselmis chui could improve SR and SL, respectively. This conclusion was consistent with Luo et al. (2004); in addition, larvae would have good performance only when fed with the mixed diets of Chlorella vulgaris or Tetraselmis chui with Pseudoisochrysis paradoxa.
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Effect of larval density on larval growth and survival Previous studies indicated the negative effects of intraspecific density on growth of bivalve larvae (Ibarra et al. 1997; Liu et al. 2006, 2010; Raghavan and Gopinathan 2008; Velasco and Barros 2008) and juveniles of snail (Baur and Baur 1992). There was a similar relationship between density and growth rate of R. venosa larva. The reason why growth rate was reduced under high density is mainly due to intraspecific competition for food and space (Liu et al. 2006, 2010; Raghavan and Gopinathan 2008; Velasco and Barros 2008). In the present study, algal concentration was adjusted according to density of larva. Such adjustment could ensure larvae to receive an equal diet in each treatment. Therefore, the limiting factor in the present study would not be food but space. The fact that the space limited the growth rate of larvae may be due to an increase of collision and chemical suppressants such as fecal waste discarded food material and mucus (Avila et al. 1997; Liu et al. 2006). Collision, which increased with the increase of rearing density, causes an abrupt cessation of beating of the velum cillia, followed by rapid retraction of the velum, and then closure of the valves. Pelagic larvae use the velum to swim and feed; thus, feeding activity was inhibited and energy expenditure was increased (Liu et al. 2006). On the other hand, previous studies showed that the accumulation of fecal waste and discarded food material increased with an increase of rearing density, leading to microbiological problems and buildup of ammonium in the tanks (Velasco and Barros 2008). However, in the present study, seawater in all cultures was exchanged every day to eliminate waste. Therefore, the accumulation of fecal waste and discarded food material may not be the main factor which reduced larval growth rate. Moreover, Avila et al. (1997) indicated that mucus strings of larvae become entangled under high density. Thus, the effect of mucus could not be excluded in our study and should be further studied. The present study showed that there was no correlation between survival rates and density unless density is over a certain level. This finding was inconsistent with most of previous reports (Ibarra et al. 1997; Liu et al. 2006) which indicated that there was no relationship between SR and density. Indeed, the density influence on larval survival is more complex than on growth rate. And, the mechanisms of survival decreased need further research.
Conclusion This study identifies a number of optimal rearing conditions for culture of larvae of Rapana venosa. The optimal temperature ranged from 28 to 31 °C at a salinity of 30 ppt. Optimal salinities ranged from 20 to 25 at a temperature of 25 °C. The mixture diet (Pseudoisochrysis paradoxa + Chlorella vulgaris + Tetraselmis chui (1:1:1)) was the appropriate diet. The optimal stocking density was ≤300 larvae/l in the initial stages and 100 larvae/l starting 10 days. Acknowledgements The research was supported by the Project supported by the National Natural Science Foundation of China (Grant No. 31572636), the NSFC-Shandong Joint Fund for Marine Science Research Centers (Grant No. U1606404), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11020703), and the Agricultural Major Application Technology Innovation Project of Shandong Province. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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