Chinese Journal of Oceanology and Limnology Vol. 32 No. 4, P. 773-782, 2014 http://dx.doi.org/10.1007/s00343-014-3264-6
Effects of temperature and salinity on the development of embryos and larvae of the veined rapa whelk Rapana venosa (Valenciennes, 1846)* BAN Shaojun (班绍君)1, 2, ZHANG Tao (张涛)1, **, PAN Hengqian (潘恒倩)3, PAN Yang (潘洋)1, 2, WANG Pingchuan (王平川)4, XUE Dongxiu (薛东秀)1, 2 1
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
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University of Chinese Academy of Sciences, Beijing 100049, China
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Ocean University of China, Qingdao 266100, China
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Qingdao Agricultural University, Qingdao 266109, China
Received Oct. 11, 2013; accepted in principle Nov. 12, 2013; accepted for publication Dec. 4, 2013 © Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2014
Abstract The major population of the veined rapa whelk Rapana venosa (Valenciennes, 1846), which is an important fishery resource, is facing a large decline in China. We studied the effects of incubation temperature (16–34°C at salinity 30) and salinity (5–45 at 25°C) on the incubation period and subsequent larval development. In the temperature experiment, the shortest incubation period was 12 days at 34°C, the lower temperature limit was 16°C, the longest mean shell length (1 193±17 μm) occurred at 25°C and the highest survival rate 72.28%±5.62% was observed at 28°C. In the salinity experiment, the shortest incubation period was 15 days at 25. The salinity tolerance range was 15–40, the longest mean shell length (855±9 μm) and the highest survival rate 72.93%±4.85% were both observed at 35. This study demonstrated that, during the egg–mass stage, temperature and salinity regimes influence later growth and survival of larvae. These observations deepen our understanding of the ecology and conservation of natural populations of Rapana venosa. Keyword: Rapana venosa; embryo; larvae; temperature; salinity
1 INTRODUCTION Environmental factors play an important role in the growth and survival of aquatic animals (Tang et al., 2012; Wang et al., 2012) and, among them, temperature and salinity are considered to be the most significant physical factors (Ahn et al., 2012; Wang et al., 2012). Temperature modifies energy flow, which regulates the rate of biological processes (Scheltema, 1967). Ambient salinity imposes an additional metabolic load, which may decrease the efficiency of metabolic processes (Bao and You, 2004). Several studies have evaluated the effects of temperature and salinity on growth and survival of bivalves, including Mytilus edulis (Brenko and Calabrese, 1969), Ostrea edulis (Robert et al., 1988), and Pinctada martensii (Wang et al., 2012). However, few studies have examined the long-term effects of temperature and salinity on gastropod mollusks (Spight, 1975; Tang et
al., 2012). The veined rapa whelk, Rapana venosa (Gastropoda, Muricidae) (Fig.1) is native to temperate Asian waters (Kool, 1993). It invaded the Black Sea in the 1940s (Drapkin, 1963) and has spread throughout the Mediterranean (Zolotarev, 1996; Harding and Mann, 2005) and, more recently, Chesapeake Bay, Virginia (Harding and Mann, 1999). Records also indicate that this species has spread to the coasts of northwest Europe, Brittany (Camus, 2001), the Netherlands (Nieweg et al., 2005), and South America (Giberto et al., 2006). * Supported by the National Key Technology Research and Development Program of China (Nos. 2011BAD13B01, 2011BAD45B01), the Chinese Academy of Science and Government Cooperation Program (No. Y12319101L), and the National Marine Public Welfare Research Program of China (No. 201305043) ** Corresponding author:
[email protected]
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Fig.1 Three developmental stages of R. venosa a. broodstock; b. egg masses; c. larvae. N 41° 40° Dalian
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Rapana venosa is a significant species, both ecologically and economically. Within its native distribution range, it is an important and valuable fishery resource but it has been overexploited and its habitat has been destroyed, resulting in severe population declines (Wei et al., 1999; Yang et al., 2008). However, within regions beyond its native range, R. venosa is regarded as an undesirable alien species because of its predation on economically important bivalve mollusks (e.g., oysters, mussels, and clams). For this reason, the basic biology of R. venosa has been studied in detail (Mann and Harding, 2003; Harding and Mann, 2005; Nieweg et al., 2005). Li (1959) and Wei et al. (1999) studied the development of larvae. Mann and Harding (2003) and Wang
et al. (2003) studied the effect of salinity on embryonic and larval development. Unfortunately, little knowledge is available concerning the effects of differing temperature and salinity regimes applied continuously during the incubation period on the subsequent growth and survival of larvae. Egg masses are attached to hard substrates (Mann et al., 2004). Conditions in the surrounding environment, such as temperature and salinity, might affect the quality of the egg mass, which could in turn influence larval growth and survival. The larval phase is the most vulnerable stage in the life cycle of the rapa whelk, as in most marine gastropods (Pechenik, 1986; Harding, 2006). The culture of egg masses, a key factor for larval rearing, is critical for increasing the population of R. venosa. Thus, more research is needed to determine the optimal conditions for rearing of egg masses. In this paper, we have studied the effects of temperature and salinity during incubation on the incubation period, embryo size, larval growth, and survival of R. venosa. This research improves our understanding of the larval ecology of this species and provides basic data for the conservation and restocking of natural populations.
2 MATERIAL AND METHOD 2.1 Source of broodstock Broodstock were obtained from Haizhou Bay (119°21′53″E/35°05′55″N–119°29′45″E/34°45′25″N), Rizhao, China, in August 2012 (Fig.2) and were conditioned in the laboratory at an ambient salinity of 30 with gradually increasing temperatures from 13°C to 25°C, for several weeks prior to spawning. The rapa whelks were held in 1-m3 tanks, which were
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supplied with flowing seawater and fed with clams Ruditapes philippinarum. The egg masses, which typically were attached to the walls of the holding tank, were collected within 24 h after deposition. 2.2 Experimental protocol 2.2.1 Experimental design The main purpose of our study was to investigate whether differing temperatures and salinities applied to egg masses during incubation affect the subsequent growth and survival rates of larvae. Egg masses from the same broodstock were spawned at 25°C and a salinity of 30, and acclimated to a series of conditions. After the larvae had hatched from egg masses, they were acclimated at 25°C and salinity of 30. All larvae were cultured at 25°C, which was found to be the optimal temperature for growth in preliminary tests. A salinity of 30 was used to be consistent with their natural oceanic habitat. The seawater used for experiments was initially filtered through gravel, sand and a sieve. Low-salinity solutions (5, 10, 15, 20, and 25) were made up by diluting seawater (30) with fresh water obtained from the city supply and aerated for at least 24 h before use. The high-salinity solutions (35, 40, and 45) were made up by adding sea salt into the seawater. The salinity of the experimental solutions was checked using YSI-556MPS. Thermostatically-controlled heaters were used to maintain the temperature of seawater to within 0.5°C. All culture tanks were refreshed daily with seawater at the same temperature and salinity to eliminate excretion products. Veliger larvae were fed daily with a mixed diet of the algae Pseudoisochrysis paradoxa, Chlorella vulgaris, and Teraselmis chui. To maintain the salinity at a constant level during experiments, the algal food was added before the required adjustment for a constant temperature and salinity. 2.2.2 The effects of temperature on incubation period and embryo size Egg masses were spawned at 25°C and salinity of 30. Temperature was then adjusted from 25°C to the experimental temperatures (16, 19, 22, 25, 28, 31, and 34°C) at a rate not exceeding 3°C/d. Three replicates were established for each treatment level and four egg masses were introduced into each replicate. Once hatched, the incubation period of each treatment was recorded and the larvae were collected by a 235-μmmesh screen within 24 h. About 2 000 larvae were
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collected from each replicate and resuspended in their original temperature conditions (25°C) at a density of 200 veligers/L. The treatments were named T1, T2, T3, T4, T5, T6, and T7 corresponding to the above series of incubation temperatures. Larvae were acclimated to these conditions by adjusting the temperature from the original temperature back to 25°C at a rate not exceeding 3°C/d at a salinity 30. 2.2.3 Effects of salinity the development of the embryo and the larvae Egg masses were spawned at 25°C and salinity of 30. Salinity was then adjusted to the experimental levels (5, 10, 15, 20, 25, 30, 35, 40, and 45) at a rate not exceeding 5/d, for egg-mass development. Three replicates were established for each of the nine salinities and four egg masses were introduced into each replicate. Once embryos hatched, the incubation period was recorded. However, egg masses did not hatch successfully at salinities of 5 and 45. Larvae were collected by a 235-μm-mesh screen with within 24 h. About 2 000 larvae were collected from each replicate and resuspended at their original salinity conditions (30) at a density of 200 veligers/L. Those treatments were successively named S1, S2, S3, S4, S5, S6, and S7 corresponding to the above series of incubation salinities (excluding 5 and 45). Larvae were acclimated to these conditions by adjusting the salinity back to 30 at a rate not exceeding 5/d at 25°C. 2.3 Sampling methods and statistical analysis In both the temperature and the salinity experiments, larvae were sampled to record shell length (the maximum dimension), shell height (perpendicular to shell length) and growth rates at 0, 5, 10, 15, and 20 days after hatching. The larvae in each culture were collected with a screen and transferred to petri dishes. After draining the seawater in the petri dishes, the larvae stopped swimming and the shell length and height were measured under a microscope with the precision of 1 μm. Thirty larvae were measured in each culture, after which the larvae were returned to their original culture. The mean shell length (SL) and mean shell height (SH) of larvae from three replicate cultures were estimated. The mean daily growth rate of larvae (GR) in each 5-day interval was calculated using the formula: GR=(SLt–SLt–5)/5, where t is the time elapsed since hatching, SL is the mean shell length.
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SR=(Dt×V)/(Dt0×V)×100, where t is the time elapsed since hatching, t0 is the hatching time and V is the volume of seawater in the culture tank. One-way ANOVA was used to test the effect of incubation salinity and incubation temperature on the development of embryos and larvae of R. venosa, and differences between means were compared using the least significant difference (LSD) test with a 95% confidence interval.
3 RESULT 3.1 The effects of temperature on incubation period and embryo size In the temperature experiment, the incubation period decreased progressively with increasing temperature. Egg masses hatched in all cultures, and no temperature limit for embryonic incubation was observed. The longest incubation period (34 days) was observed at 16°C, and the shortest was 12 days at 34°C. The rate of decrease in incubation period decreased as temperature increased; the largest decrease was observed between 22°C and 25°C (Fig.3). At temperatures higher than 25°C, differences
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Fig.3 Effects of temperature on the incubation period of R. venosa
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Fig.4 Mean shell length of R. venosa after incubation and hatching at different temperatures Bars are SEM. Means with the same symbol are not significantly different (P>0.05).
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In this study, the larval density (D) was used to represent the effect of incubation temperature and salinity on the survival rate. To estimate the survival rate, the seawater within which the larvae were kept was mixed to create an even distribution of larvae and a cup of seawater was sampled to estimate the population density. After counting the number of larvae and measuring the volume of seawater acquired, the larval density, expressed as the number of larvae/L, was calculated. The above procedure was repeated three times to ensure the accuracy of the result, and the survival rate (SR) was estimated by the following formula:
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Fig.5 Mean shell height of R. venosa after incubation and hatching at different temperatures Bars are SEM. Means with same symbol are not significantly different (P>0.01).
in the incubation period were not statistically significant (P>0.05). Temperature had a significant effect on the SL (F(6,203)=58.149, P
